Modulation of PAK1 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of PAK1. The compositions comprise oligonucleotides, targeted to nucleic acid encoding PAK1. Methods of using these compounds for modulation of PAK1 expression and for diagnosis and treatment of disease associated with expression of PAK1 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of PAK1. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding PAK1. Such compounds are shown herein to modulate the expression of PAK1.

BACKGROUND OF THE INVENTION

[0002] The Rho family of GTPases (Rho, Rac, and Cdc42) are Rasrelated small GTP-binding proteins (p21^(ras) proteins) that regulate coordinated changes in the actin cytoskeleton and the formation of distinct cytoskeletal structures. Controlled changes to the actin cytoskeleton are vital for many cellular processes, including motility, adhesion, cell division, cell death and phagocytosis. Mediating the signals of the Rho GTPases Cdc42 and Rac is a family of serine/threonine p21-activated kinases (PAKs). The PAK family comprises at least four isoforms, PAK1, PAK2, PAK3 and PAK4, which are critical reorganization but are also involved in nuclear signaling. The PAK proteins have been implicated in a wide range of biological activities such as neurite formation and axonal guidance, development of cell polarity and motile responses (Daniels and Bokoch, Trends Biochem. Sci., 1999, 24, 350-355).

[0003] One of these PAKs, PAK1, specifically activates the JNK1 MAP Kinase signaling pathway in mammals and can also function in place of Ste20, the analogous yeast protein which activates the yeast pheromone response MAP Kinase cascade and regulates cellular morphogenesis. The gene encoding PAK1 (also called p21-activated kinase 1, PAK-alpha, alpha-PAK, hPAK1, yeast Ste20-related, and CDC42/RAC1 effector) was cloned in 1996 and the resulting 545-amino acid protein is 98% identical to Rat PAK1 (Brown et al., Curr. Biol., 1996, 6, 598-605). The chromosomal location of the PAK1 gene is at 11q13.5 to q14, a region which is commonly amplified in a subset of estrogen receptor positive breast carcinomas prone to metastasis (Bekri et al., Cytogenet. Cell Genet., 1997, 79, 125-131).

[0004] PAK1 has been implicated in the progression of breast cancer cells. Etk/Bmx, a nonreceptor protein-tyrosine kinase that controls the proliferation of mammary epithelial cancer cells, directly associates with and phosphorylates PAK1 (Bagheri-Yarmand et al., J. Biol. Chem., 2001, 276, 29403-29409). Furthermore, the expression of kinase-active mutant PAK1 in breast cancer cells stimulates anchorage-independent growth (Vadlamudi et al., J. Biol. Chem., 2000, 275, 3623836244).

[0005] Recent studies in platelets have provided evidence that PAK1 may be an important physiological regulator of scavenger receptor class B, type I (SR-BI), a high density lipoprotein receptor that mediates the flux of cholesterol between high density lipoprotein and cells. SR-BI overexpression in mice reduces atherosclerosis, which may be a manifestation of SRBI's ability to enhance hepatic uptake of high density lipoprotein cholesterol. The PAK1 pathway has been shown to downregulate the SR-BI promoter, thus PAK1 may play a role in cholesterol homeostasis in the atherosclerotic vessel wall (Hullinger et al., J. Biol. Chem., 2001, 276, 46807-46814).

[0006] The perturbation of normal cell survival mechanisms leading to increased cell survival plays an important role in the development of a number of disease states. A critical regulatory component of the cell death pathway is Bad, a protein whose pro-apoptotic effects are directly inhibited by phosphorylation by PAK1. Thus, the important part that PAK1 plays in promoting cell survival pathways has been suggested to be a relevant mechanism for promoting diseases in which the apoptotic responsiveness is reduced, including cancer (Schurmann et al., Mol. Cell. Biol., 2000, 20, 453-461). The phosphorylation of Bad by PAK1 is also stimulated by Nef proteins encoded by human immunodeficiency virus, and Nef anti-apoptotic effects are likely a crucial mechanism for HIV replication and thus AIDS pathogenesis (Wolf et al., Nat. Med., 2001, 7, 1217-1224).

[0007] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PAK1 and to date, investigative strategies aimed at elucidating PAK1 function and mechanism of activation have involved the use of antibodies and inactive mutants.

[0008] An antibody to the autophosphorylated, activated form of PAK1 was used to reveal the spatial and temporal distribution of activated PAK1 in fibroblasts (Sells et al., J. Cell Biol., 2000, 151, 1449-1458).

[0009] Mutations in the small G protein Ras, a key regulator of cellular proliferation and differentiation, are commonly found in tumors. A mechanism through which Ras induces cellular transformation occurs via the JNK signaling cascade, and PAK1 has been postulated to be an effector of this pathway. A catalytically inactive PAK1 mutant inhibits Ras transformation of rat-1 fibroblasts, suggesting that PAK1 is involved in Ras-induced cellular transformation (Tang et al., Mol. Cell. Biol., 1997, 17, 4454-4464).

[0010] PAK1 contains a regulatory domain and a kinase catalytic domain which can interact intramolecularly resulting in a closed, inactive configuration (Tu and Wigler, Mol. Cell. Biol., 1999, 19, 602-611). This autoinhibition is decreased in PAK1 containing mutations in the regulatory region (Frost et al., J. Biol. Chem., 1998, 273, 28191-28198). Mutants of PAK1 have been generated in several places including amino terminal mutants, kinase dead mutants all with the intention of discovering the mechanism through which Cdc42, Rac1, and Raf are able to activate PAK1 or uncovering the resulting effect on cellular motility and actin organization (Frost et al., J. Biol. Chem., 1998, 273, 28191-28198; Sells et al., J. Cell Biol., 1999, 145, 837-849; Sells et al., Curr. Biol., 1997, 7, 202-210; Zang et al., J. Biol. Chem., 2002, 277, 4395-4405). A non-phosphorylatable mutant of PAK1 lacking threonine 212 was expressed in neurons to demonstrate dramatic neurite disorganization (Rashid et al., J. Biol. Chem., 2001, 276, 49043-49052).

[0011] Consequently, there remains a long felt need for agents capable of effectively inhibiting PAK1 function.

[0012] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PAK1 expression.

[0013] The present invention provides compositions and methods for modulating PAK1 expression.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding PAK1, and which modulate the expression of PAK1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of PAK1 and methods of modulating the expression of PAK1 in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of PAK1 are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0015] A. Overview of the Invention

[0016] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding PAK1. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding PAK1. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding PAK1” have been used for convenience to encompass DNA encoding PAK1, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0017] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of PAK1. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0018] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0019] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0020] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0021] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0022] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0023] B. Compounds of the Invention

[0024] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0025] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0026] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0027] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0028] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0029] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0030] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0031] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0032] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0033] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0034] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0035] C. Targets of the Invention

[0036] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes PAK1.

[0037] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0038] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding PAK1, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0039] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0040] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0041] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0042] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0043] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0044] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0045] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0046] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0047] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0048] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0049] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0050] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0051] D. Screening and Target Validation

[0052] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of PAK1. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding PAK1 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding PAK1 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding PAK1. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding PAK1, the modulator may then be employed in further investigative studies of the function of PAK1, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0053] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0054] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0055] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between PAK1 and a disease state, phenotype, or condition. These methods include detecting or modulating PAK1 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of PAK1 and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0056] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0057] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0058] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0059] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0060] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0061] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding PAK1. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective PAK1 inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding PAK1 and in the amplification of said nucleic acid molecules for detection or for use in further studies of PAK1. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding PAK1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of PAK1 in a sample may also be prepared.

[0062] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0063] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of PAK1 is treated by Administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a PAK1 inhibitor. The PAK1 inhibitors of the present invention effectively inhibit the activity of the PAK1 protein or inhibit the expression of the PAK1 protein. In one embodiment, the activity or expression of PAK1 in an animal is inhibited by about 10%. Preferably, the activity or expression of PAK1 in an animal is inhibited by about 30%. More preferably, the activity or expression of PAK1 in an animal is inhibited by 50% or more.

[0064] For example, the reduction of the expression of PAK1 may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding PAK1 protein and/or the PAK1 protein itself.

[0065] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0066] F. Modifications

[0067] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0068] Modified Internucleoside Linkages (Backbones)

[0069] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0070] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-51 linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 31 to 3′, 51 to 5′ or 2′ to 21 linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0071] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0072] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0073] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0074] Modified Sugar and Internucleoside Linkages-Mimetics

[0075] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0076] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0077] Modified Sugars

[0078] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a 0 (CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0079] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0080] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0081] Natural and Modified Nucleobases

[0082] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-amino-adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0083] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0084] Conjugates

[0085] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed October 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0086] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0087] Chimeric Compounds

[0088] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0089] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0090] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0091] G. Formulations

[0092] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295, 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,530,575; and 5,595;756, each of which is herein incorporated by reference.

[0093] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0094] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0095] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0096] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0097] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0098] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0099] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0100] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0102] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0103] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0104] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0105] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0106] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0107] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0108] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0109] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0110] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0111] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0112] H. Dosing

[0113] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0114] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0115] Synthesis of Nucleoside Phosphoramidites

[0116] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0117] Oligonucleotide and Oligonucleoside Synthesis

[0118] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0119] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0120] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0121] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0122] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0123] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0124] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0125] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0126] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0127] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0128] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0129] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0130] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0131] RNA Synthesis

[0132] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0133] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0134] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0135] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0136] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0137] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedron Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0138] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5×annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0139] Synthesis of Chimeric Oligonucleotides

[0140] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0141] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0142] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 51 and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0143] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides

[0144] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0145] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0146] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0147] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0148] Design and Screening of Duplexed Antisense Compounds Targeting PAK1

[0149] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target PAK1. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0150] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0151] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5×solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0152] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate PAK1 expression.

[0153] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 uL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0154] Oligonucleotide Isolation

[0155] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0156] Oligonucleotide Synthesis—96 Well Plate Format

[0157] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0158] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0159] Oligonucleotide Analysis—96-Well Plate Format

[0160] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0161] Cell Culture and Oligonucleotide Treatment

[0162] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0163] T-24 Cells:

[0164] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0165] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0166] A549 Cells:

[0167] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0168] NHDF Cells:

[0169] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0170] HEK Cells:

[0171] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0172] Treatment with Antisense Compounds:

[0173] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0174] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0175] Analysis of Oligonucleotide Inhibition of PAK1 Expression

[0176] Antisense modulation of PAK1 expression can be assayed in a variety of ways known in the art. For example, PAK1 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0177] Protein levels of PAK1 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to PAK1 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0178] Design of Phenotypic Assays and In Vivo Studies for the Use of PAK1 Inhibitors

[0179] Phenotypic Assays

[0180] Once PAK1 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.

[0181] Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of PAK1 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0182] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with PAK1 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0183] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0184] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the PAK1 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0185] In Vivo Studies

[0186] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0187] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or PAK1 inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a PAK1 inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0188] Volunteers receive either the PAK1 inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding PAK1 or PAK1 protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0189] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0190] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and PAK1 inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the PAK1 inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0191] RNA Isolation

[0192] Poly(A)+ mRNA Isolation

[0193] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0194] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0195] Total RNA Isolation

[0196] Total RNA was isolated using an RNEASY96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0197] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0198] Real-Time Quantitative PCR Analysis of PAK1 mRNA Levels

[0199] Quantitation of PAK1 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0200] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0201] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 r2M of probe, 4 Units RNAse inhibitor 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0202] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0203] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0204] Probes and primers to human PAK1 were designed to hybridize to a human PAK1 sequence, using published sequence information (residues 1686266 to 1756308 of the sequence with GenBank accession number NT_(—)030106.2, incorporated herein as SEQ ID NO: 4). For human PAK1 the PCR primers were: forward primer: TGTGATTGAACCACTTCCTGTCA (SEQ ID NO: 5) reverse primer: GGAGTGGTGTTATTTTCAGTAGGTGAA (SEQ ID NO: 6) and the PCR probe was: FAM-TCCAACTCGGGACGTGGCTACA TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0205] Northern Blot Analysis of PAK1 mRNA Levels

[0206] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0207] To detect human PAK1, a human PAK1 specific probe was prepared by PCR using the forward primer TGTGATTGAACCACTTCCTGTCA (SEQ ID NO: 5) and the reverse primer GGAGTGGTGTTATTTTCAGTAGGTGAA (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldelhyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0208] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0209] Antisense Inhibition of Human PAK1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0210] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human PAK1 RNA, using published sequences (residues 1686266 to 1756308 of the sequence with GenBank accession number NT_(—)030106.2, incorporated herein as SEQ ID NO: 4, GenBank accession number AL042444.2, incorporated herein as SEQ ID NO: 11, GenBank accession number F29045.1, incorporated herein as SEQ ID NO: 13, the complement of the sequence with GenBank accession number AI650866.1, incorporated herein as SEQ ID NO: 14, and GenBank accession number U24152.1, incorporated herein as SEQ ID NO: 16). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human PAK1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human PAK1 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO NO 232136 5′UTR 15 111 atggcctctgaggcaggagg 55 17 1 232137 start 4 205 ttgtcaccaccagcagcagc 76 18 1 codon 232138 start 4 210 tgacattgtcaccaccagca 91 19 1 codon 232139 start 4 215 ttatttgacattgtcaccac 70 20 1 codon 232140 exon 4 342 ctctgggtttggaggcagag 78 21 1 232141 exon 4 347 ttctcctctgggtttggagg 82 22 1 232142 exon 4 12828 aaactcccctgtgacagcat 79 23 1 232143 Coding 15 667 cccgtaaactcccctgtgac 78 24 1 232144 Coding 15 672 gcattcccgtaaactcccct 95 25 1 232145 exon 4 13368 tgaagcaagcgggcccactg 85 26 1 232146 exon 4 13429 caacacatccagaacagcct 76 27 1 232147 Coding 15 826 tcagctgacttatctgtaaa 79 28 1 232149 Coding 15 861 ccttcacattcaaggcatta 68 29 1 232150 exon 4 33811 gggcgtggagcaatcactgg 74 30 1 232152 exon 4 36914 ggaagtggttcaatcacaga 84 31 1 232155 exon 4 36998 ttccgggtcaaagcatctgg 95 32 1 232156 exon 4 37003 cagtattccgggtcaaagca 96 33 1 232157 exon 4 37032 agacattttaggcttcttct 94 34 1 232158 exon 4 37038 ctcatcagacattttaggct 95 35 1 232159 exon 4 39148 atcgcccacactcactatgc 85 36 1 232160 exon 4 39153 ttaggatcgcccacactcac 87 37 1 232161 exon 4 39158 tcttcttaggatcgcccaca 88 38 1 232162 exon 4 48850 attaataatcagctctttct 81 39 1 232163 exon 4 48856 gatctcattaataatcagct 90 40 1 232164 exon 15 1382 cacgaggtaactgtccaagt 85 41 1 232165 exon 4 51992 aacaacccacagctcatctc 77 42 1 232166 exon 4 51998 ttccataacaacccacagct 72 43 1 232167 exon 4 52022 tgtcaaggagcctccagcca 95 44 1 232168 exon 4 52027 acatctgtcaaggagcctcc 89 45 1 232169 exon 4 52032 tcaccacatctgtcaaggag 90 46 1 232170 exon 4 59936 gaaagtcccggaagatagct 82 47 1 232171 exon 4 59980 agctgaacctctcttctcca 87 48 1 232172 exon 4 59986 ctctttagctgaacctctct 83 49 1 232173 Coding 15 1942 atcttcaggaattgatgctg 77 50 1 232174 exon 4 69390 ggcttggcaatcttcaggaa 85 51 1 232175 exon 4 69423 gctgcagcaatcagtggagt 80 52 1 232176 3′UTR 15 2048 agagcttggcacaatgaggc 88 53 1 232177 exon 4 69525 aggagttggaatttctgaaa 80 54 1 232178 exon 4 69563 aggaaatgggagaagcaagg 84 55 1 232179 exon 4 69568 agatcaggaaatgggagaag 70 56 1 232180 exon 4 69575 gagtgctagatcaggaaatg 74 57 1 232181 exon 4 69584 agtcttgaggagtgctagat 88 58 1 232182 exon 4 69597 ttccaaggatcaaagtcttg 77 59 1 232183 exon 4 69612 tgctggacacacggtttcca 83 60 1 232184 exon 4 69657 aaatggccatcatctgatta 78 61 1 232185 exon 4 69666 cttatttagaaatggccatc 92 62 1 232186 exon 4 69681 attgggaggaaattccttat 90 63 1 232187 exon 4 69696 ccctcatatccatgaattgg 85 64 1 232188 exon 4 69736 ctagaaacatttatttatat 56 65 1 232189 5′UTR 11 190 accgcctagttcactggctc 0 66 1 232190 5′UTR 11 197 aggcctgaccgcctagttca 41 67 1 232191 5′UTR 11 358 agagcctgtgagggaagcgc 11 68 1 232192 exon 4 69800 tatagtcaagaattaattgt 38 69 1 232193 genomic 13 127 ccagcagcagctactggtgg 71 70 1 232194 genomic 14 99 tagtgctggtatttgacatt 61 71 1 232196 exon 4 69849 gaaatctcaattgattacaa 62 73 1 232197 exon 4 69934 aaccccatggcattcccaag 74 74 1 232198 exon 4 69971 ggaagctgagcctcttcatg 73 75 1 232199 exon 4 69992 ctgagccaaagtcatggtcc 70 76 1 232200 exon 4 70012 ttctcccatgtcaggatcag 80 77 1 232210 intron 4 2300 ttctgacttcaggtgatcca 86 87 1 232211 intron 4 35776 cacaattatcttttgcatag 84 88 1 232212 intron: 4 39135 actatgcttcctttgaaaga 55 89 1 exon junction 232213 intron 4 39902 catatagcctaggtgtgtag 89 90 1 232214 intron: 4 43447 gcctgaagcactgaacagta 93 91 1 exon junction 232215 intron: 4 48803 taatggccacctgaaatcaa 82 92 1 exon junction 232216 intron 4 49316 ctcaaatgaaacatctagtt 84 93 1 232217 intron: 4 55411 tcctacttacttagcttgac 60 94 1 exon junction

[0211] As shown in Table 1, SEQ ID NOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93 and 94 demonstrated at least 50% inhibition of human PAK1 expression in this assay and are therefore preferred. More preferred are SEQ ID NOs 44, 91 and 62. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target segments identified in PAK1. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 148691 15 111 cctcctgcctcagaggccat 17 H. sapiens 95 148692 4 375 gctgctgctggtggtgacaa 18 H. sapiens 96 148693 4 380 tgctggtggtgacaatgtca 19 H. sapiens 97 148694 4 385 gtggtgacaatgtcaaataa 20 H. sapiens 98 148695 4 512 ctctgcctccaaacccagag 21 H. sapiens 99 148696 4 517 cctccaaacccagaggagaa 22 H. sapiens 100 148697 4 662 atgctgtcacaggggagttt 23 H. sapiens 101 148698 15 667 gtcacaggggagtttacggg 24 H. sapiens 102 148699 15 672 aggggagtttacgggaatgc 25 H. sapiens 103 148700 4 697 cagtgggcccgcttgcttca 26 H. sapiens 104 148701 4 758 aggctgttctggatgtgttg 27 H. sapiens 105 148702 15 826 tttacagataagtcagctga 28 H. sapiens 106 148703 15 861 taatgccttgaatgtgaagg 29 H. sapiens 107 148704 4 955 ccagtgattgctccacgccc 30 H. sapiens 108 148705 4 1003 tctgtgattgaaccacttcc 31 H. sapiens 109 148706 4 1087 ccagatgctttgacccggaa 32 H. sapiens 110 148707 4 1092 tgctttgacccggaatactg 33 H. sapiens 111 148708 4 1121 agaagaagcctaaaatgtct 34 H. sapiens 112 148709 4 1127 agcctaaaatgtctgatgag 35 H. sapiens 113 148710 4 1169 gcatagtgagtgtgggcgat 36 H. sapiens 114 148711 4 1174 gtgagtgtgggcgatcctaa 37 H. sapiens 115 148712 4 1179 tgtgggcgatcctaagaaga 38 H. sapiens 116 148713 4 1316 agaaagagctgattattaat 39 H. sapiens 117 148714 4 1322 agctgattattaatgagatc 40 H. sapiens 118 148715 15 1382 acttggacagttacctcgtg 41 H. sapiens 119 148716 4 1403 gagatgagctgtgggttgtt 42 H. sapiens 120 148717 4 1409 agctgtgggttgttatggaa 43 H. sapiens 121 148718 4 1433 tggctggaggctccttgaca 44 H. sapiens 122 148719 4 1438 ggaggctccttgacagatgt 45 H. sapiens 123 148720 4 1443 ctccttgacagatgtggtga 46 H. sapiens 124 148721 4 1866 agctatcttccgggactttc 47 H. sapiens 125 148722 4 1910 tggagaagagaggttcagct 48 H. sapiens 126 148723 4 1916 agagaggttcagctaaagag 49 H. sapiens 127 148724 15 1942 cagcatcaattcctgaagat 50 H. sapiens 128 148725 4 1951 ttcctgaagattgccaagcc 51 H. sapiens 129 148726 4 1984 actccactgattgctgcagc 52 H. sapiens 130 148727 15 2048 gcctcattgtgccaagctct 53 H. sapiens 131 148728 4 2084 tttcagaaattccaactcct 54 H. sapiens 132 148729 4 2122 ccttgcttctcccatttcct 55 H. sapiens 133 148730 4 2127 cttctcccatttcctgatct 56 H. sapiens 134 148731 4 2134 catttcctgatctagcactc 57 H. sapiens 135 148732 4 2143 atctagcactcctcaagact 58 H. sapiens 136 148733 4 2156 caagactttgatccttggaa 59 H. sapiens 137 148734 4 2171 tggaaaccgtgtgtccagca 60 H. sapiens 138 148735 4 2216 taatcagatgatggccattt 61 H. sapiens 139 148736 4 2225 gatggccatttctaaataag 62 H. sapiens 140 148737 4 2240 ataaggaatttcctcccaat 63 H. sapiens 141 148738 4 2255 ccaattcatggatatgaggg 64 H. sapiens 142 148739 4 2295 atataaataaatgtttctag 65 H. sapiens 143 148744 13 127 ccaccagtagctgctgctgg 70 H. sapiens 144 148745 14 99 aatgtcaaataccagcacta 71 H. sapiens 145 148747 4 2156 ttgtaatcaattgagatttc 73 H. sapiens 147 148748 4 2241 cttgggaatgccatggggtt 74 H. sapiens 148 148749 4 2278 catgaagaggctcagcttcc 75 H. sapiens 149 148750 4 2299 ggaccatgactttggctcag 76 H. sapiens 150 148751 4 2319 ctgatcctgacatgggagaa 77 H. sapiens 151 148761 4 2300 tggatcacctgaagtcagaa 87 H. sapiens 160 148762 4 35776 ctatgcaaaagataattgtg 88 H. sapiens 161 148763 4 39135 tctttcaaaggaagcatagt 89 H. sapiens 162 148764 4 39902 ctacacacctaggctatatg 90 H. sapiens 163 148765 4 43447 tactgttcagtgcttcaggc 91 H. sapiens 164 148766 4 48803 ttgatttcaggtggccatta 92 H. sapiens 165 148767 4 49316 aactagatgtttcatttgag 93 H. sapiens 166 148768 4 55411 gtcaagctaagtaagtagga 94 H. sapiens 167

[0212] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of PAK1.

[0213] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0214] Western Blot Analysis of PAK1 Protein Levels

[0215] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to PAK1 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 167 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 3 atgcattctg cccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 70043 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 4 ttgtgatttt tcattagtgc ttggcatata gtaggcattt acatcagtga gtgttagcaa 60 tatcatggtg tgcagtggaa ggacttgagc tagaccctga aggaagggct gtataatttt 120 gattctttgc aattttttaa catgtatttc cttttggtca aaaataataa aagagttata 180 ttgttcttat ttctgattct agtagctgct gctggtggtg acaatgtcaa ataacggcct 240 agacattcaa gacaaacccc cagcccctcc gatgagaaat accagcacta tgattggagc 300 cggcagcaaa gatgctggaa ccctaaacca tggttctaaa cctctgcctc caaacccaga 360 ggagaagaaa aagaaggacc gattttaccg atccatttta cctggagata aaagtatagt 420 atttgatttt cttctcatca tttatatgct ttaaaaaaat tattttgggt aacctggata 480 aaagatctgt tgtctttgac ttaattttcc tttaatgttt tctttctggt taaatatttc 540 cctttctccc tacagtggct gaagcaaaat ggaaaataga cctattttta tactctatta 600 actatatctg agcaaaactg taagagaata attatgctct tgccagattt gggacaatct 660 gaagtgattt atttattttt aaattcttaa actgtttatg aagccacaaa atgtaagaaa 720 aattgaaatg tctttatttg gaattttttt ccccccgagt tagtttcaat tggaaatgtt 780 ttctttccaa aatcttgatt gccttttctt tattccagct tcctggtccc actttcaggt 840 ttggggacta cgtatcagta agaatggcaa gtaacagctg agaaaaggat gcagagataa 900 ctagcattca gcatgagttt actatgaata aattatcctg ggcagcctca tttccttttt 960 attgataaaa cttctgttaa tagaagagta atcatatgat gaatagtggt gattttaaca 1020 ctgattttaa cactgttgat ttaatagtgt atatgaaaaa gtcaccaaga tcctcgtaga 1080 caaggttgta aaataaaggt tttggtgtcc acaacaaaag atgacttttg tttggtacca 1140 aaaatgttga tttaagtggc tgttgctctg aagtaaagtt ctccttatat ttgtgggccc 1200 tgttcttatg gcatgctaaa gatactggct ttctgtgtgt aagtagttac catgagcttc 1260 tggtttagat ggagctgggt caccacttac cagctttctg atcttaggca tgtcagttaa 1320 ctgctttgac ctttatttag tttcctattt taaaagtgag atatcatttc acagggttgt 1380 tgtgaagact acatacaaag ttcctaatag agtaccttcc acacagaagg tatttgaatg 1440 ttagttctct gtctctttac tcctttgtct ctgtctcttt actcctttgt tgatgaatag 1500 aaactgggag gcatagctgg tagactacat ggtaaaatca gaactgagat tctgaaacag 1560 tagggcagat acaacaagca caatggtaga ttgcggaaaa tatctcatgt tgtagaaaaa 1620 tataattgcc taactacgga tcaggggaga tggtagatgg tagtgtcatt agcccaatag 1680 ggaacataga aagaaggaac aaattaggag tgttggtgat tggagagcag ggaagatgat 1740 tattttggac acattgcatt tgtagtgtct ttatatagat atgtcttata agcagatctc 1800 atgtattgta aatgtcgtac aagtgaaatg agaaataata aggactttta atgagtacat 1860 tgggcttata tcgttatgtg tatgagtcct tctacttcac cttcacttcc ctttgggttt 1920 ggtggctttg ggtagaaaag aaggccaggc acaggggctc atgcctgtaa tcccaacact 1980 ttgggaggcc gaggtgggag gattgcttga gctcaggagt ttgagactag cccgggcaaa 2040 ataggaagac cttgtctcta ctaaaaaaaa aaaaaaaaaa ggagtcgggc atggtggcgt 2100 gtgcctgtag taacagctgc ttgggaggct gaggcaggag gattgcttga gcctgggaat 2160 ttgaatctgc agtgagctgt gatcatgcca ctgcactcca gcctgggtga cagagtgaga 2220 tcctgtctta aaaaaataaa ataggccggg tgtggtggct catgcctgta atcccagcac 2280 tttgggaggc tgaggcgggt ggatcacctg aagtcagaag ttcaagatca gcctggccaa 2340 catggtgaaa ccccatctct actaaaatac aaaaaatcag ctgggcatgc tggcaggcgc 2400 ctataatccc agctactcag gaggctgagg ctgggagtat cacttgaacc cgggaggcgg 2460 aggttgcagt gagccaaggt tgcgccattg cactccagcc taggcaacaa gagctaaact 2520 ccatctcaca aaaaataaat aaataaaata aaagatatgg gtaaggtgct taaccttgat 2580 ttcctgacta attcattcat atagtagctg ccaacttcct tggtacaagg gctatgatct 2640 gagacagtgt taaatgccct gcagtcttct tccacatatg tagccacttt gtttatccct 2700 caatttagat gtgtatttat ttttaatatt gtatcttatc aaccagtgtt cttgatctct 2760 ataaataggc tttcagccca agtgtctaca tgtagtgaaa gaggactttt taaattaaaa 2820 agtttaaatt gttcttgctg actcaaacta gaaaggtaat aagattctca tccagtaggc 2880 atcatcaaaa ctaaaaacat ttatgtttca aagggcatca tccggaaagc aaaaaggtaa 2940 cacacagaaa ggagaaaaat ttcccaaatt acatatttta taatggatgt gtatctaaaa 3000 tatgtgaaga agaactattg caagtcaaca accgaaagac aagtaatttt aaaatgggca 3060 aaatatctaa atagacatat ctccaaagaa catatacaaa tggccaagaa gcacatgaaa 3120 ggattctcaa atcattagcc atcagggaaa tgcaaatcaa aaccacaatg agataccact 3180 tcacactcac taggatggct ataataaaaa agataataac aagtgttagt gaggatgtgg 3240 agaaattgca accctcataa ttgctggtgg gaatgtaaaa tggtgcagcc tctgtggaaa 3300 acagtctggc agttcctcaa aagattaaac atagagttac cagcagtaca tgaatgttta 3360 tagcagcatt cataatagcc aaaaagcaga aacaacccaa ataccccatc agctgatgaa 3420 tagataaata ataaaatgtg gaatatccat acaatgaaat atttggtaac aaagacatga 3480 agttctgatc cgtgccatga catggatgac ccttgaaaat gctgtgttaa gtgaaagaag 3540 ccagttacaa aggatcacat atagtatgat tctatttgta ggaaatgtcc agcattggta 3600 aattcataga tagaaagtag attagcggtt gccttgggct gcagagaggg agaggcaatt 3660 aaaactagga gatgattact aagaggggtg gggtttcttt ttgtagtgat gaaatgttct 3720 aaaattgatt ttggtgatac ctgtccagct ctctgaatat attaaaagcc attgaatttt 3780 acacttcatg ggtgaattgt ttggtaagtg aattacatct caataaagct gatttttaaa 3840 aaagaagatt ccagtctgaa acagcagatg gaagcagagc cttcagacat cattggaacc 3900 tggagggaca atcttatgtg ctaaatggga aagtgggggt ctagatattg ggattatagt 3960 attggttctt ccataaattg gctgtctgat ttgggtcaat tatttctctg tagacgtcag 4020 tgccttcatc tagaaaagga gagagagcag taggcatgtg cagtttcttt tagctttaga 4080 atcctataac tggccaggca cggtggctca tgcctgtaat cccaccactt tgggaggccg 4140 aggcgggtgg atcacgaggt caggagatgg agaccatcct ggctaacacg gtgaaacccc 4200 gtctctacta gaaatacaaa aaataagccg ggcgtggtgg cgggtgcctg tagtcccagc 4260 tgctggggag gctgaggcag gagaatggcg tgaacccagg aggcggagct tgcagtgagc 4320 caagattgcg ccactgcact tcagcctggg cgacagagcg agactccgtc tcaaaaaaca 4380 aaataaaata aaatagaatc ctataattac ataaatgtga tgttgcaaag gcgtggttag 4440 atcaggcttg tctatatagg tgaaaagaaa ctaggccatt gtaaagtact ttattcaatg 4500 atacagctgt agaaaatcct aggctaaatc taagtaacct taggctaaat ctgatctgaa 4560 aatacatctc atagcctcaa gatcaaaact gcatctctgc aggctgaaaa catagaggta 4620 gaaaacatac agagttataa gaagttcttg caaaatgatc taactatctt tcatcctttg 4680 gttacagatt ttctatattt gccctgggca cataggtttc atccctttgc tttaagaccc 4740 ttgatggcta taactaatgc atacatcaaa atgaaagaag tctgaggtgt ttttcttttg 4800 tttggagcct ttgatcaacc agtaagctac tcaacttagt cctataagga tacacagcag 4860 tgttggtagt ctttgaagtg gtatatgccc ttcatgtagt atttgagcat ttataccagc 4920 tgcatagagt tatgtctgga actggactta taacacagat tgagtccagc cactgacatt 4980 tgttaactct gatttatttt ttaaatttca ttccaatatc ctcacttccc attcatccta 5040 tatcattagt aggcttttgt catttgacat agtagatata aaagcatttt ctaaactaaa 5100 atgcaataca aataggaggt ttgtttttta aaaatggaaa cctcatttat tgttttattt 5160 gatacctaca acaactcatt tttagttggg gaggctaagg cttagagaaa agtaagttgc 5220 tcaaggtctc acaagtggaa agtgggagag ctgagtatgg aactaagtct tcctaaatta 5280 tctgttccat tgtaacataa ctatctctct tagttgttct caaccctgat tacatgttgg 5340 actcacttat ggtgctttta aaaaatacag ggcggggtgc aagtggctca cgcctgtaac 5400 cccagaagtt tgggaggccg aggcgggcgg atcacttgag gtcaggagtt ctagaccagc 5460 ctggccaaca tggcaaaacc ctgtctctac taaaaataca gaaattagcc ggttgtggtg 5520 gcaggcacct gtaatcccaa ctactcagga ggctgaggca ggagaatcgc ttgaatctgg 5580 gcggcagagg ttgcagtgag ccaagatcat gctactgcac tccagcctgg gtaacagagt 5640 gaaactccat tacaaaaaaa aaaaaaaaaa ggctgggcgt ggtggctcac acctttaacc 5700 ccagcacttt gggaggccaa ggcgggtgga tcacttgagg ccaggagctc aagactagcc 5760 tggccaacat ggtgaaaccc tgtgtctact aaaaatacaa aaattagctg gacgtggtag 5820 tgggcacctg taattccagc tacacagaag gctgaggcag gagaattgct tgaacccagg 5880 aggcagaggt tgcagtgagc tgagatcaca ccactgtact ccagcatggg tgacagagca 5940 aaattctgtc tcaaaaataa ttaattaatt aattaatact gatacctgca ttccacccag 6000 acctttggag tcagaatttc tagaggagtt gtcttgggtc tctagagaga tttttgggag 6060 atgagaaaga agaatgatct gtaggctgta gtaatggttg agatttgagc tgccagtggg 6120 tagaatgtct ttgggcatta cctaatgctg acctctctag catggcctca gtatgtgcct 6180 gggggatgca taaatgattg gaaagagaag ctgtaattga gggcctgtaa taggtgatgc 6240 tgagttgcag aattccatgt gggaaagcct ggttcctgtg gttaggatct ggagcctttg 6300 gtttattcta agaagtcttt cagcatttag tttgaaatct ttttctggct gcattttcac 6360 aagcaaagtg gaattctctg agcttgggaa gagtttagca aagttaattt taacattgga 6420 ctatgtcagt gtttatctct ccaagtttgg aacgttttac tgccttgagc tcagattagc 6480 tcagtggagc caatgagagg tgctgcttaa aaatcatttt tttaaaaaag gccagtgatt 6540 tgtgctgaaa ctttgatggc tataccaaga catgggattt tgcagtgtca gcctcatttc 6600 ccatgctatc agaaagccaa gtggtctagg tcaaagatag ggttaaagtt gggatgggtg 6660 aggttggaag cttcttttcc cgctttattt tctcaatccc aagatctcta gatacatgaa 6720 atcaagatga ttctttcctc tttctcacct caaccacagt ttctctgtct taatgaatgg 6780 gctgagaaac ctaacagttt tttatattaa tgacaactca tgtttgtaaa gttctttagt 6840 ttacaaagca catatatttc ctgtaactta aagaattctt ttttttttga gacagtcacg 6900 ctctgttgtc cgggctagag tgcagtggcg cgatctcagc tcactgcaac ctccacctcc 6960 tgggttcaag caattctccc tgcctcagcc tcccgagtag ctgggattat aggcacctgc 7020 caccacgcct ggctaatttt tgtatttttt agtagaggcg gggtttcacc atgttggcca 7080 ggctggtctt gaactcctga cctcaggtga ttcacccgcc acggctgccc aaagtgctgg 7140 gattacaggc atgggccact gcgcccagcc aagaattctt aacatcgttg taggaatcct 7200 tatttagata taagtcttct gtcagttata tatgttacaa atatcttctg tgctcttagc 7260 ttttattaga ttaagtaaat tcccctgtaa tcttagacag atgcttggca tgatgtgaga 7320 tagaatttta atgtggtcaa atttattaat tatttccttt ctaatgcttt gtggttctat 7380 ttttgaaatc tttacctact cagagacatg aaggtttttc tattgagttt tctgtattca 7440 cgtacatgag cacatgatct gtaaattgta atagttcttt ttctttcttt gtaatccttc 7500 tccttttact tctttttctt gcctaattgc actggttaat accttcagta ctatgatgaa 7560 tagaagtggt gaggaggaat acccttctcc tgtttttaat ctcaggaaaa gctttcagca 7620 ttttagaatt gagtataatg tttgctccag tttttggtaa ttacctttta ttagattaag 7680 taaattccct tgtaatccta gtttgctaag agtttctaat catgattgat atcacatttc 7740 agcaaatgct tttttctgca ttaattgaat tgattatata tattttcttc tttattttct 7800 taatatagta aattacattc actttttttt aaaaaattga gatataattc acatgccata 7860 caagtcacca tcttaaaatg tacaattccg tggtttttag tatattcata agattgtaca 7920 accattacaa ctaattccaa tacattttca tcactccaga aagaaacctc atactcattg 7980 cagtcgttcc tcacttctcc cttctcccct ggcaaccact aatctactct gtgtctatgg 8040 atttgcctat tctggacatt tcatataaat ggaattacac agtatattgc cctttgtgac 8100 tggcttcttt cacttagcat aatgttttca aggttcatcc atgttgtagc atggatcagg 8160 acttcatttc tcttcatggc tgaataatag tatatggatg tactgcattt tatctattcg 8220 tcacttgagg gacatttggg ttgccttcac tttttgactg ttatgaataa gctaatgtga 8280 acattcacat acaagttttt gtgtagacat aagttttcat cagattcctg cagacatact 8340 tctagtcatc ataaaaaatt tgcaactgca gcagtagtcc cagggatact atatccctgg 8400 gatatgggac catcaggatc ctaggtttgc tttattttct ccattgtgtc ttccctcagt 8460 ccgaagctct tagtgccttc ctgcctgcag tcatgtgctt tgtgaatctg gtaagagttt 8520 atgaaatttg gttgacttgc tgagcaggtg gaaagatgga agaaagatca cactgcttac 8580 ttcttttggc ctctgcttat tctgtttctt ttggaaatgc cctttcccat ctcggtctgg 8640 ctaacccctg ttcatccttt atgacttctt tcaggtatta tcttctccag aagagtggtt 8700 tattacttac cttcttctgt acacagagca tgcaccattg tcctattttg cagattgaag 8760 acttgaaact cagactttcc ctgggtcacc cagtgagtta ctggcaaaaa taatactgaa 8820 tccttcctct gagattcttt ccactaacag aaatttcttt taattccctg acctcagctg 8880 tttagctcct gggtttgtgg tgatgagcca gtgagacagt gactggcaga gcttggtgaa 8940 gggctgtaca caggtgaagg ggatgattgt tacttccaag aagggattaa ttgggttcca 9000 ggtggctctg ggactgtatg acttctccat ttctctcctg tattcctgcc tcttgaatcc 9060 tgttttctgc tggtgatccc caactggtga atactcagtc taaatattac acagtaccag 9120 gccgggcgtg gtggctcatg cctgtaatac cagcactttg ggaggccaag gtgggtggat 9180 cacccgagtt caggagttcg agaccagcct ggcaacatgg tgaaaccctg tctctactgc 9240 aaatacaaca agtaactggg tatggtggcg ggtgcctgta atctcagcta cttgggaggc 9300 tgaggcaggg agaattgctt gaaccgggga ggtggaggtt gcagtgagct gagatcacgc 9360 cattgtactc cagcctgggc aacaagagtg agactctgtc tcaaaaaaaa aaaaaaaaaa 9420 aattacacag tacccaagct aaatcctgcc acctagattt ccccgttcct tttacttctc 9480 tcctctccca cttcaaccaa gtcattgcta ttcttgtact ccgcgtctaa tctatgttaa 9540 actgttattt cagttagtag ttgataccag tcaaatctca gtagggatca ggggtataat 9600 aaagagtgaa agacttgatc aggtagacta cagtcctgtg ctctgtgaat caggacaagt 9660 caacttacat ccctgggcct ctttcctcag ggaatgggat catggtctct agagtcactc 9720 cctaatattt tctttcttta tttttttgag acacagcctc actctgtcac ccaggctgga 9780 gtgcagtggt acaatttcag ttcacttcaa cctctgcctc ctggttcaag ctattctcat 9840 gcttcagcct cctgagtagc tgggattaca cgcatgtgtc tccacgccca gctaattttt 9900 gcatttttag tagagatgga gtttcaccat gttggccagg ctggtcttga actcctgacc 9960 tcaagtgatc cgcctgcttc agcctcccaa agtgctggga ttacagacgt gagccaccgt 10020 gcccggccat tccctaatat tttctagagt gttcttcaac taagtaagcc atgtgctgta 10080 ttgaaattca gaagtacagg gattgtaatt ggaagtggta attttggtcc tcaagaagct 10140 tacattttgg atttttaaac tagcaaggaa ttatataaga taggaaatga ctagatgctt 10200 ggtggtgtag cagactcagt gtcttgggaa ttaacaggaa gaagaaaggt caggaaatcc 10260 ttcctggaaa aagtgggacc tgagccagtc tttaaaagat tgctgagata gggagaagga 10320 gaagagagca ttccaaacaa ggagaagaaa aagcaagcaa aaagttggag gtgggaatat 10380 gtgaggtata ttcaaaggtt gcagggtaaa gagtgctcag tagaagcaga gctgacttta 10440 gcattaccct gtgtgttcat gtattaaatg atttcatttc tctggaactc ttttctgtaa 10500 accactcatg ctttgagcta actttctttt tgggtctcct ttaccaaagt ttcttatgca 10560 tttgacagtt catgcctgtg atgtattata tagtatatca attatatata atagaaataa 10620 tgtataaaca tgcatagata tagaaaataa aaattgaaat aaaacacaaa tataattgta 10680 cttccctaag tgactagatc agagggaatt tagaaacaga aagtagttcc aaagccctgc 10740 cacatgttcc agtgcattct ggctatgaga aagacagatg gacttacgtc ataaaggact 10800 gcttgacttg gggccctggt atacccctta gccttctctt tctcaaagca gctgcttctg 10860 agagggaaat tgccaagtgc ataaatagct taaaccctag cacttggcat gtaggcaaat 10920 ggttctcatt aagatactga agaatgaacg tttggaatca gacctgggtt caaatcccag 10980 ctctcagtag ctgtatgagt aaattactca gtttcctcat ctgtaaaatg acagtagtag 11040 tatttatctc ataggattgt aagaatgacg gtagtaatat ctatcttgta ggattgtaag 11100 aatgaagaaa aatactatat tgacatcatg gtttgtatac atggcaagtt cttaatgtca 11160 tttcttctta ctacactgaa gtatgttttc ttgattcaac ttgaaatttt ttaggggagg 11220 taaaatagaa tgtgaagagc cttggctttg atgccagact ttttggattt taattactta 11280 atgattgtag gcaagtctgc tcccaaattt tctaatctac aagtaaaact tacctcagga 11340 ttgtggtgat tgtattagat gatatgtatg aaatacctat ttaaaactgt gggatgccat 11400 ctcaggaagg aatcttaact gcagagcttt gagaaagcat agtttcttgt tttcatagat 11460 tcacagaatg taggtgcttg agggaccctt aagatcatct agctcatcct ctcccttgtt 11520 tagatgagaa agcagagatc ctggtctctc tttctctctc tctctctaca cacacacaca 11580 cacacacaca cacacacaca cacactctgt ggtgaggtag aggtgaaact ggcttgaact 11640 ctagaccttt ttgttttttc cttctcattt tgctaccttt tatcagggtt ttgctgggtc 11700 tgtggaggtg ttagaacttc agaatttctt ttggttagtg tgttgagctc ccagacagca 11760 gggttttaaa ataaggttca caggtgctgg gtaacaagtg ttgtgaaagt caggccagag 11820 ctgctgaaat gaacatttat tcctgccctg gagagaattt ttgtgtctta ggcagattct 11880 ttaaactcct tgcccagttg agttaaatgt ctggtcattt tttcctttct tcacttactg 11940 tctgagcacc tactttgtcc aggtgtgtgc tagcctctgg gaagctgtga ttaatttgac 12000 atggcctctg cacttagaat tcagtctggt atggaagata aacacttata atagtcaatt 12060 actattacat atattgggtg ctgtgggaac agagagggta aagaaccacc cagcctagag 12120 atcaaggagg gcttcacaaa aaagatgact tactctatat caactggcta gttactggta 12180 aaaccagaac tgttaactct gacagacgat ctttttgcct ctccctttat tttattgttt 12240 tttgtttttt tgagacaggg cctcactctg tcacccaggc tggaatgcag tggcgtgatc 12300 tcagctcact gcaacctcta catcctgggc tcaaatgatc ctcttgcctc agcttcacaa 12360 gtagctggca ttacaggcat gcaccaccat gtctagctaa tttttgtatt tttgcagaga 12420 cagggttttg ccatgttgcc taggctggtc ttgaactcct ggactcaagc aatctgcctg 12480 ccttggcctc ccaaagtgct gggattacag gcatgaacca ccgtgcccgg ccacctcccg 12540 tactttagtt caacaagaaa ttaagtcttt aaacatgcat tgagtatctg ctccaatcca 12600 gatccgtaac tgagctcttc tacatatgtg aaatattaat agatagactt tgccctctca 12660 aggagactac agctcaataa agagctaaaa taagcctaaa aacaataatg ttcctgtttt 12720 ttatttcctt ttgcctgttt ctcttgcagc aaataaaaag aaagagaaag agcggccaga 12780 gatttctctc ccttcagatt ttgaacacac aattcatgtc ggttttgatg ctgtcacagg 12840 ggagtttacg gtaagtcctg ggtcacagaa aggtctttag tcttttagag ttctggctgt 12900 gcactgttag attcaaccct ttctcatctt cctgatgctt ctatctttgc ctgagaccct 12960 gattacaatt ttctcacgtt catgagtggg acactttcat cgccaaaatt gtatccagag 13020 aagcctttcc atctccgttc cttaaataat aaacctctgt taaatggtag gcttggtagc 13080 aggctctgtg tggggcactg atcatgcagg aacaacatcc tatcctacca tcctggaggt 13140 atagcctggt gaaggagcag acatcttaag aaatgactaa ttcaatgaga aagatattat 13200 aaaagaggct tgtgtaaact ctgagagtat agagagaaag gaatgactaa taaatgcatc 13260 ttttgctgct agcaagtgtc aaagttagct ctgatgttct ttcaggctcc cctgtgactg 13320 tgccttcctg ttagtcttgc atgtctctct tacagggaat gccagagcag tgggcccgct 13380 tgcttcagac atcaaatatc actaagtcgg agcagaagaa aaacccgcag gctgttctgg 13440 atgtgttgga gttttacaac tcgaagaaga catccaacag ccagaaatac atgagcttta 13500 caggtatgag aactgcgtcc agggcagtct cagcaagaga tggttttaat ctgggtggga 13560 aagctcacat gcctattagt ctgggatgag agaagaaagt agtggaaaga gtggaagtgg 13620 cactttaacg tggaattttg gaatttgaca gccaagatga tggactttgc atcaaacttg 13680 agtcttcact ctgtcactgc acacgttttt ttaaaaatag catggtatta tatagagaga 13740 acacaggttt tatgctagaa ggtctagatt ctgaccttta ctaggttcta ccagtcactt 13800 catttctttg catctcaaaa tgggaattcc tatctcaaga ggctgtagtg agaaatggtt 13860 gggcgcagtg cctcacgcct gtaatcccaa cactttggga ggccaaggca agaggatcgc 13920 ttgaggtcag gagttcgaga ccagtctggg caacgtggca aaaccctgtc tctacaaaaa 13980 atacaaaaat tagccgggtg tggtggtgca tccctgtggt cccagctact tgggaagctg 14040 aggagaaggg atcacttgag cctgggaggt ggagttgcag tgagcaagga tcacaccact 14100 acactccagc ctgggcaaca gagtgagacc ctgtctcaaa aaaaagaggt tatagtgaga 14160 aataagtgag ctgattgatg tgagagttct ttataagttg taaaacatca ccatagaaat 14220 gtagctatca ttgtcatcat tattgttaat attattatta gttatttaat aacattaatg 14280 tatgtcttcc tcagggaact aatttttatt agttaaatga aaaacaggcc aataaagaat 14340 aagtttcttt agatgtaccc tcggtagaag ggactatatt tttcagagtg tttttaatag 14400 cagaattggg attagaaccc aggtctccta attcctgtaa catcaccttt tgaatttact 14460 acttgcccct aataatgtta tttggaggct ctccttccac agcggaccag ctttccattg 14520 ttttgcaggt gtgagaacca aagaaggcct ttctaatatg ctaaattaat agtgggctaa 14580 tcttttaatt tctaggctct ttactcatgt tgcctccttc aaatggcagt attgttggga 14640 aggtgagaac caggatgaac tagtgaattt tgaaggatgc tttccccagc cagtatctgg 14700 cttgtttcaa gggcacagat ggtggagatg acaggctgca gcttcactca tgctgctatg 14760 gtggtgccca ggagagggcc atgcatgtgc ccagcagtat ttccttcctc tcgtctggcc 14820 ccgctgagtc tgtctcactt actccattga gggactgagt catttcaagg aggaattggg 14880 ctggagggct actgagtggg aaagttttct agtcaggaaa ttagagtttg tgtttaagga 14940 acttgtcatg gaaacatcag gttagaggct gtctttgaaa acctcagtgg ccaagtcttt 15000 gcaaggatct ttcctgcatg tatgtatgta aatatgtgct agtggcatgt aaatgtgtat 15060 gtaaacagag acatagccct gtgggtttgc atatatttgg aattaaagcc tacttaagta 15120 ttcatgaaca aagataaagt ccactcaggt attcatgaac gaacatatgt ataaatataa 15180 acattcaggt aattatgtgt atgttcattc atgcatgcac atatgaataa tattgagtgt 15240 atatgtacag atacgtgtgt acatctactt ttgtatgtat aaatttgtgc acatgtatat 15300 atatgtatgc aagaatttta attttctata agttgaagac tagggccaac tctaggcaaa 15360 attgcatatt tggtataggt ttttttaagt gtcatatctc tcctttccct atttaccatc 15420 ctcttatact attagttatg ataatactac ctcatttgta tatgctcaca tgtgaattac 15480 ctcatgtgat tcttttgctg gagtcctgtg gtgtgatctt ggctcactgc agcctgtgcc 15540 tcctgggctc aggtgattct cctgcctcag cctcctgagt aactgggact gcaggcatgc 15600 accaccaccc ccagctaatt tttgtgtttt aatagagaca gttcaccatg ttggccaggc 15660 tggtctggaa ctcctgacct caagtgacct gctcaccttg gcttcccaca gttctgggat 15720 tactggtgtg agccactgag cctggctccc tcatgtgatt cttatgatgg ccttagattg 15780 gcagggcaag gactattatg ctcattttat atactcaaag aaattaatta ggctcagaga 15840 agttaaatga cataccccaa gtgactcagc tgatacatga tggaaatgta ttttaagttg 15900 ggcctttaac tcctttgctg cctataaccg aagcacaatc ttccatgcta atttttttgt 15960 caagtatttc tattttttca acacttaatt tttttttttt tttttttgag atggagtctc 16020 actctgtcac ccaggctgga gtgcagcagc gcgatctcgg ctcactgcaa catctgcctc 16080 ctgggttcat gcaattcttc tgcctcagcc tcccaggtag ctgggactac aggtgcatgc 16140 cactatgcct ggctaatttt tgtatttgta gtagagatgg ggtttcacca tattggccag 16200 gctggtcttg acctattgac ctcatgatcc acccaccttg gcctcccaaa gtgctgggat 16260 tacaggcgtg agccatgtgc ctggcaacac ttaatttttt atacctgtta gtagctaatg 16320 taatagggaa aaagtagact agattgacaa ctcaactggg tcttagccct attttgccac 16380 tgacttacgt gaccttgaac caatcttttt ccttttctgc acctcatttt gctcatctgt 16440 aaaatgaaag tgctggaagc actttctgtt ccatcattct gtgctttgga atttgatttt 16500 gatatataga agttcatcac ctttaaaggg agtgtttact tatttttata tgacataaca 16560 agtatgccta ttaccaaaat acccaatctt caaaaatact agcagtacgt aaagttgggg 16620 gtagtgcagt gaaggcatag tgttgtactc tgccaaagtt gatagtctat agtcttttgg 16680 acaagaaaaa acaaacgaat tatgtttaaa gtatatcctg aatttatttc tccagaatac 16740 tgctagctat tctggatttg ttataagaac aagttgtcag agcttgcctt atttcctgct 16800 ttgtgggttc atgatttgct gtagtgtcaa agaagctgta tctagcactt ttttacatga 16860 tggaattgga attgtacatt ctctgggctt ggttcttggt gaggaatata ttggatttgg 16920 tagaatattt cccctcctta attctattag tggtgaattg gcagtcagtt tttcttggac 16980 ccaggggaat taaagaacaa aactgaagac attacctggt ctttaaactt actttcttcc 17040 taagatctcc attgggtctc tctttcaaat tttatgaagt ctaagatacc actgattgta 17100 agatgtacaa ttatttgcat accaatcaaa gaaaataaag ctgccaatta aaccataaca 17160 tgctttagaa agtaggacac acctctgttt cagagacgtt aatgtgaaaa aatgtacatc 17220 tcagaatcag tgaaacatgg taattgcttc attgagggag cttgattcta ttcttggaaa 17280 acaagaccct acctttaatt agacttgaca ggatcaatac ctctcttttc ctcaacctta 17340 gtccttttgt ccttgtgggc agtagatatg gatatagaag gatcctttat gaaagctgaa 17400 actggagact ggccatgttt tggcatggat gactcaggat ggtgctatgg attctttcct 17460 tttcttttag aatgcagatt ttcttggcca tttaagcctg acagtgatat agctgatgat 17520 gtggatacag ttctagcgta gtatttagaa tgagaccaga attcaaatct aattcctctc 17580 tttaacctgt gtgtaccctg ggcaaataac agtcatgtgc cacaacaaca gaccgcatat 17640 acaatggtgg tctgataatt aatattataa gggagttgaa aaattcttat cacctagtga 17700 ttcctgaagt ttaaaataaa tttagcatag cctaagtgta cactggtaat gtcctactta 17760 ggccttcaca tttactcatc actcactcac tgactcaccc agagcaactt ccagtcctgc 17820 aggctctatt tatggtaagt gctcatatag gtgtatcctt ttatttacct tgtaaaccat 17880 attttttatt gtaccttttc tatgtttaga tatacaaata cttaccgttg tgttacagtt 17940 gcctgcagta acgtgctgta caggtttgta gcctagaaag caataggcta tacaatatag 18000 cccaggtatg tagcaggcta taccgtctag gtttgtgtaa gtacactcta tgatgttcac 18060 ataatgacca aattacctga tgacacattt cacagaacat accccattgc taagccacac 18120 ccaactgtac ttaacatctc tgaacttgtt tccaaatcaa taaaagtaga tataataata 18180 gtactcagag ccataatgag gattaaagaa ggtgatatgt ataatgcatt tagcatgata 18240 tctagcctgt agtaccagtc atactattac tattctctga atctggcacg tgcttgacca 18300 gatagacttc ttttgctgtc cagaaaaatt taactgtaac tgttattgac taagtccctt 18360 gtttttcttc ctgtgcagat aagtcagctg aggattacaa ttcttctaat gccttggtaa 18420 gtctttattt actctatttt gatgtgggca gatgtggagg taaggatcaa gacagtgtgg 18480 tagaggcaga gcctgagctg ggaggcactc atattagtca ctcctgcacc cttggcctca 18540 gaattcctat ctatccaata aagaaattag atataataaa tagggggtat ccctttcagc 18600 tttaatgtcc tagaaattct ttctttgtga taattttttt atttttaatt tttatggatt 18660 cataatagtc gtaaaatgtt ctagaaattc taattttgga accagaaggt atggctttag 18720 actcagtttt aatttgtagc tttgaacctt gggaaagtca gtttgcctct ttgagcttca 18780 atttcttcat caataaaatg aggatgatac actgtgccct cacctggaca tgtcttcaga 18840 tacctacact tagaagaaaa tttcctcttt ggtctatgga gtctgcaaaa ccaagattag 18900 tactaattgt tgaagggagc agattttatt catttaaagt cattcattct gtcctctgaa 18960 gttgtaatga atgctttgac actcaaatgg atgttataga aatcatgaag aattggctgg 19020 agagtaaact agatgatctc tctgtctctc ttaacttaca aattctattt cccagggcac 19080 ttacagggat aaaatgtgat gttgatgtat ttgaaagggt cctataaata gtaaaatgtg 19140 atgcatataa gagaatactg cttttttttt tttttttttt taaagagacg aggttttgct 19200 ctgttgccca ggcgggagtg caatggtgtg atcatagctc agtgtaaact caaactcctg 19260 ggctcaaatg atgctcctgc cccagccctc tgaatagctg ggactacagg tataagccat 19320 cacactcagc ttattgagtg tgatgtcaaa ttgtcagaca gacaattttt taaaagtttt 19380 ggtagagatg tggttctcat tatgtgttct caaactcctg gcttcaagtg atcctcctgc 19440 ctcgacctcc caaagtgtta ggattacagg tgtgagccac cacacctggt tgctatttat 19500 tttttacatc tgatgttgtc tcttgtacat aggtggttat gccatattga tttactaaac 19560 atttgcaaaa gccctagttt atcctatgcc aagtccggtg tgaataagtg acattacaga 19620 gatggtcaga catgtcccag tacctaagaa cctcccagtc ttgaggttga agacctcaag 19680 cagccagttc ttcatgattc ctgtttgtgc ttgaaggcac agacagacaa ttatagtgca 19740 gcatgctaac cttcatgaag ccaggggctg tctctgactt gttaattgtt atatccttgg 19800 catctggcaa gtcttggcat ttggtaaaca tttactgaat gaaaggttgg atgtgtcaga 19860 gaaagcttcc tgatggtaaa gtctgagctg aatgagttag ttatctagag cagggattaa 19920 ggggaaatgc gggtattcca cacagaaaga gaatagcatg tatgtgggca aggaagttca 19980 tgaggcatac ttgcacaggg aactgcgaag tggttatgac tagaatgtga atagtgaagg 20040 aagtagaggt gggtgatgag gaggattggt aagcaggaca gatcatgtag gagtttgact 20100 tatcttaaag gtgatgagac gcttaaagaa cttaagcaga agagtggtta agttctaaca 20160 caataccgag tcaccatcaa aatactttga accatgcttc caccagtggg gactggttta 20220 ttcggagtca tttttacaca tattgattat aaaaaacaat attcacacgt tcagtgcctt 20280 gcaacctgtt ggatcctttc ttactgactc tctgaatcct cacaggagct tactgaggta 20340 agttattatc cttgtagatg ataaaactga aatggaaaag agttaatggc aataggcatc 20400 tgattcccag cctatactgc tttcccctga tttgtgctgc ctttcatcaa atagaagaga 20460 tgtgattaaa agatgggaaa gaatttgcaa atataggatc tttgaatagt aaattattaa 20520 agaagaggct ttaagccgaa ttgtgctttg tggctcaaat gtctttatgc ttttgtagga 20580 agagtgatag ctatcaatat tgagtgcctg cgatgcacgg tgttaaaaac tttatgtaag 20640 catgattagc ctcacaatta cttatgtaca aagtattgtc atctccattt cataggcaga 20700 aaaacagagt ttcctgaaga cttataatta gaaccacatt acaactgagt cttgtgaatc 20760 tcagtttagg gttcttttca ctgtggcaag gaacaaaaaa agatgtcatt gaggtcttat 20820 aaaggagact gcagtaaagc tgtagctatg ttgtttgtta ttgaggagga agagccctgg 20880 ggttcatgcc atatggcagg gaggataaca cctgcttggt aaaaacttgt actggtttag 20940 tatctacaat taaaaccact aaggctgtca aggacattgt tctcccctag ttcagaacat 21000 ttgtccagca ctagctcagt gctctatata tccttactag ttttacatgg aggatatgtt 21060 ttgtattatg gtagcatgtc attattttcc atgttggttt caaacagatg attccacttg 21120 tgaaataatc aaatataatt gctgcctaag aaattaagac tcagaaagat taagtgactc 21180 actccctacg tttgaatgag ttagtagtag aaaccagcac aaaattctgc tctcctgagc 21240 attcatgtat tcactcatac cctaaatatt gacttagtat cttctttatg ccaaaataca 21300 agtgacacaa aaatgaacgt gcactgtctt aatccttgag aaaaaaaatt aatgacagac 21360 tggggagata agggctgcaa gagaggcatg aataatgtac aatagggtta tatttgatgg 21420 agtgatttat tctgctctga agtacaagga agtttgcaga gttcttgggc cttaaagctt 21480 tgttcgttta acaaacataa tcttaaagtc ccttttagct ctaaattctg ttatctgtgg 21540 ttcactggtc tgagatctga ctctaagatt ccatggcatt ttaatgcctg attctcattt 21600 ataaaatgaa tagttaccac atgataagtt ctttgggtgc tgagaagtca aggactagta 21660 agatctctgc tcttggtctc acaatttttg tgatgaatca gaacatgtaa acaaggattt 21720 ataatatagt gttaagtaag catcaggtaa tgataccagg cagagaaaaa ggcagtgctt 21780 tcctgtctga tggacagcat gaacaaaagt tgggttaagg atccagtatg ttaggccaga 21840 gagcttggac tttgtgctgg agctgaggga ctattttaag tagaagattg atgtagttgg 21900 atatgtgatg tctaaactaa catcttagat ttctttggaa attggatcaa gatagtaaac 21960 tgaattcatg tgcccatctc tcttgctatc tcaaatttct taatttcaga aaagatttct 22020 taaaaaccaa attaaaagca atgctctgaa acaaaagcat gacataaatc aactgttgtg 22080 aggaattcat ggaagataga aagcagaaaa ggtcagattg atcagagcag aagaagccac 22140 agcttaaagt atgtgtaaga gaagatgcct aggaaaatga aagtagctcc agagaaaatg 22200 ggctgaaaga atatatacaa gaagcaggaa agagtcatgg gtcatcttca agggtaaatt 22260 aatttaattc tacctttgaa acagtagggg cagcctcctc cttacagtga acaaaaggca 22320 gcaggtgcat ttgattctaa gctaaaatca gctaattttc tgaagaaagt aaactgccta 22380 cttgaggaaa gctcttgctg tgggtatagg ggcctcagag agaaacagag gaacctgagt 22440 taactgcaaa attctaccgc agatataaag aaaaggaaac aggagaagta gctctgcagt 22500 gaaggaaaat acattaaagt tttccatccc aagagtaaat ttctcatact tttgatacct 22560 gggaggaggg gaatctctac ttttccatgc ctacataccc taaagtaaca cttgccaata 22620 accatctatc tataactcac aaacagatcg tatcagacct cccattcaga gtaggacagt 22680 ggaaaaacag acttacataa gtttcagaaa aactaatgct aattacatac actaatctat 22740 ccttcactta taaatatgaa tgaatagcta aggatctgca gacatttgag agaaacacaa 22800 cattaaagag aggaaccaag ataaggaagg aaaattagcc ccagaggaag tagataagtc 22860 aggaaataac ttttaaaaaa atctagtatt ttcagagagg atctgtagat gtttcatcta 22920 caaaaacacc ctgctattag agcaatcaga ggaggaaaga atgttcttag tattaaaaac 22980 ataagacata aatttatggc tgggtgcggt gctcacacct gtaatcccag cactttggga 23040 ggctgaggcg ggtggatcac ctgaggtcag gagttcgaga ccagcctggc caacatggtg 23100 aaaccccatc tctactaaaa atataaaaat tagctaggtg tgggggtata tgcctgtaat 23160 cccagctact tgggaggctg aggcaggata atcacttgaa cctgggaggc agaggttgca 23220 gtgagctgag attatgccac tgccctccaa cctgggtgac gaagcgagac tccatctcaa 23280 aaaaaaaaaa aaaaaaaaag aaatgtataa ggtttaaaca acaaatgcat ataagtgaat 23340 agggaattaa taatataaaa tataaaagat agaaaatttg agaataaatg acaatagagg 23400 gttggtcagg gggattcaag cataagcaga gaacagaggc agtagagtaa aaaaataaag 23460 aaaaaataga agaaaaaatt cccagctgaa gaaaggcatg agtcttcaga tgaagggcct 23520 gttaatgacc aagtcagata attgaaaaga gatccaagaa taaagagaaa aatcttaaaa 23580 gcctccaaag agaatatagt tcatgtccaa aggaacatta gcatcagaca tctcattaga 23640 aatagtggaa aaaacagcag tatcttcaga gggatgaggc aaattgttta gaaccaagaa 23700 ttttgtacct aatcaaacta acatttaact ataaggtgtc aatataaagg tatgtttgga 23760 catgtgaaga tcaaggttta ccacccacag atcacttctg aaaggctaaa taccaaatag 23820 taacatcaat tacctctggg gttaatggtg attaagaata ctcacttaaa agtattttta 23880 ccggccaggc gcggtggctc acgcctgtaa tacaaaaaaa ttagctgggc ttggtgttgt 23940 gtgcctgtaa tcccagctac tcaggaggcc gaggcaaaag aatcacttga accagggagg 24000 cagacgttgc agtgagccga gatcatgcca ctgcactcca gcctgggccg cagagtggga 24060 ctgtatctca aaaaaaaaat aaataaaaaa tattattatc caggtatttg attaaaaagt 24120 caagtaggac ttgtaataag tagctctcat tctgggatct tccttcacca ttgtcttggc 24180 aatccctttg catttttgtg ttggtattcc tattttttct atgccacgcc tacattattt 24240 tttgtttact tttgaatttg gtggagcata tcctctagta gctttaagaa agtaaatgga 24300 aagtaaacat ttaataatac ctttcaggtc tcaaaatggg gcgttctcta tgctaccctc 24360 acgtttaatt aatacttcag ctgcttatag aattgcaggt tacacatatt ttttcatcag 24420 aatttggaaa acattacact tttgtcatct agctttcggt actgctaagt ccaaagccat 24480 tttatttatc cttctctgta tgtgaccaat ctttttttct actcagaaag cttgtagggt 24540 cgtctttgtc accattgctt agaaatttca caattatatt cctatttaca tctatttata 24600 ctgcagggag ctttgtgaac tctttcggcc tggaaactct gccctcagtt ctaggaaatt 24660 gcttgaatta ttttgttgat gatttcttcc ccttgcttct tttgttatct cttcatggaa 24720 cctccatcat ttagatcttg gacctttttg tttgtttgtt tgttttgaga cggagtctcg 24780 ttctgtcacc aggctggagt gcagcggcgt gatctcagct cactgcaacc tccgcttccc 24840 ggttcaagca attcctctgc ctcagcttcc ctagtagctg ggactgcagg cacgcaccac 24900 catgccaggc taattttttt ttgtatatta gtagagacgg ggtttcacca tgttggccag 24960 gatgatttcg atctcctgag gttgtgatct gcccgcctca gcctcccaga gtgctgggat 25020 tacaggcgtg agccaccacg cccagcccta gatcttggac ctcttaaact aattctccat 25080 tttttaaagg attttctctc ttttccttgc gttggttttt attctgaaga attaggagac 25140 ttcctcaact tcatctttca gtcaatttaa tttttcattt ctattctcat gtttaatttt 25200 caatcacttt tttattttaa gaaaatatat agcattcttg tttccagaat tgatatattt 25260 tcttatctct aaggaaggta atgataggct ttctttggat gttttttcct tcctgtttag 25320 tctgtttttt ccaggtggcc ctttgcctca tttgttttgg tttcacactt ttatgttaga 25380 ggttttcctt gcgtattcag aggtctgctc atatttatga ttggggtagt aaaaagctca 25440 ctagaatttt aagctcattg gtggtggaca attcaatact tttaataaat ttatagagtt 25500 gtgccgccat cacctgaaaa agttccctag tgccctttta ccattcattc aaactcccat 25560 gctcagccct aggaaaccac tgatctgctt ctggtcttta taaatttgtc tttctagaca 25620 ttttatataa ataaactcat acaacatgta gtcttttgtg ttttgcttct tttacttagc 25680 tttgaacttt atgttgtaac atgtatcagt atggtgtatt tcatttcttg ttatgtccaa 25740 atcatattcc cttgtatgga tacactacat tttatttatc ttctcatcag ttgatgtgta 25800 cttgggttgt tcctactttt tggtcattat gaataatgca gttatgaaca ttcacataca 25860 aggttttgtg tggtctcatt tcttcagagt atataccaag gaatggaact gctggggcat 25920 gtggttaact ttatgtttaa ccttttgagg aactgccaga cttttttcca cagtgctggc 25980 accattttat attcccacta gcaattaatg agggttccag tttctgccta tctttgcctg 26040 tccctgttgt tgtctgtctt tttattgtag tcattttagt gggtgtgaag tggtatctca 26100 ttgtgatttt attggatttc ctaatggata atgatgttga gcatcttttc atgtgcttat 26160 tggccattta tatacaatct acttcacatt aacagtaact gaaatccagt aaaatatgta 26220 aactttgccc cagtatattt tcattccctg ccccttcttt attctattac tgccattctt 26280 tcatatacat gaatgtgtat gaatctgtat gtgtatatgt gtgtgtgcac gtgtacgtac 26340 tgtatatatt ggaagcctaa caattgcttt atacaattat gttttttaaa gaagtcaatg 26400 gaagaaataa caaaaagtat ttttttagag tcttccatgt taatctacat atttaccgtt 26460 tcctctgctc tccattatta cttgtggatt tgagttacta tttggtatta cttcgtttca 26520 gtctgatggg cttctttgag tatttcttgt aaatcaggtc tgatagcaat aaattctctt 26580 tctctttgtt aatctggaat gtctttattt cgtcttcatt ttgaaggata gttttgttgg 26640 gtatagattt cttgactggc aatttttaaa aatttcagct gtttgaatat gtcatcccac 26700 tgccttttga cctccatttt ttctgatcag aagtttttgt tttgattttt aaaatactct 26760 gatcagcagt cagctgttaa ttatactgac tggccttgta catgatgtca gttttctctg 26820 gttgctttaa atattttcat gttgacttta gcttttggca gtctgactgt gcgtttaggt 26880 gttatttttt tttttttctt tttctttttc tgagacggag tctcgctctg tcgcccaggc 26940 tggagtacag tggtgcgatc ttggttcact gcaacctctg cctcctgagt tcaggcgatt 27000 ctcttgcctc agccgcccga gtagctggga tttcaggtgc atgccaccac agccagctaa 27060 tttttgtatt tttagtagag atggggtttc accatattgg ccaggctggt cccaaactcc 27120 tgacctcaag tgatccgcct gcctcagcct cccaaagtgc tgggattaca ggcatgagcc 27180 accgctccca gccaggtgtg aatctttttg tgttttccta ctttgggatt tatcgaggtt 27240 cttggatctg tagtttaatg tttttatcaa atttaggaag ttttatcctt atttctttaa 27300 atattttttc tgtgtctttt ttttttcttc ctcagagact ctcattatat gtatgctggt 27360 gtgctttatg ttgttccaca gctctttgat gctctgtttt tttctttaat ttttttttac 27420 ctcagttctt tatattagat aatggctatg ggtccatttt cttttttctt ttttcttttt 27480 tttttttttt ttgagacaga gtcttgctct gtcacccagg ctggagtgca gtggcacaat 27540 ctcagctcac tgcaacctct gcctcctgag ttcaagcgat tctcctgcct caccctcttg 27600 agtaactggg acaacaggca cgtgccaccg cacccagcta attttatttt tatattttta 27660 gtagagatgg ggtttcacca tattggccag gctggtctcg aactcctgac ttcgtgatcc 27720 gcccaccttg gcctcccaaa gtgctaggat tacaggtgtg agccaccgct ccccacctcc 27780 attttcaaat tcattgattc ttctgccata tagaatccct tgtttagctc tctggtgagt 27840 ttttcatttg ttattttatt cttcaactgt agtatttcca tttggttcat ttgaagtaat 27900 gtctactgag aatttcactc gttgttaggc tttcttaaaa ctctttaaac atggtttctt 27960 ttagtttttt gaatgtattt atatctgagt tgaaatcttt attctctaaa gccaacattt 28020 ggggacattc agagttcctg ttgattgctt ttttccccct aggtcgtgct ttcctgtttc 28080 tttccatgtc ttataatttc ttgttgaaaa ttcaatattt agataatatt ctgtaccaat 28140 tctggatact gatagccccc tcccccaaaa aagttgctag cttttttatt tgttggtttt 28200 gtattatctt actagggata aatctgtgac atagtctctg tctgtggtca ttgatgtcat 28260 tgctcagtgt ttgttgtttt tttgtttgtt tttgtggtgg ttttgttgtt gttgttttgt 28320 tttgttgcag cctggctttc cagagattgc ttctatgttt gcttagctga atctttagcc 28380 aatgatttga cacagatggt gctcaaaaac ttcaactctg taaggcttct ttcctctgtt 28440 gatggatcta tgtgtaggta ggggagtaca tttactgttt aggccttttt caagtctgtt 28500 ccagctttta ctttatgttg ggctcttttg aatcttttat gtgtatgcac atagccaggc 28560 atgtgtgact agcatgaggc ttttctagcg tctgctgtgc ctgtgaacag ccttagccag 28620 aaattagttt gccctactca ggactgtagc ttcaggctaa tagagctgaa tagttgaccc 28680 tctgccgcta ccacctttgg aagtcacttc caatgacagt gccactgggc ctgggcattg 28740 cccatatctc cagcacaaag tgaatgttct tggactatgc agttataccc tggtctgccc 28800 tgatggaacc cccacacttc tggagttggg gctcggatgg atgggaacat cccttgccca 28860 gaatgccaga gattctattt ttaacccaaa gttcaggagt ttatcagtca taagcacttc 28920 tcaatatttt gttgaccttt gttggatttc cagggaactg aaatggtttt tgtcaatttt 28980 gtccagattt atagttgctt gttggggaga ggatttgcca gtcttctctc tctgccatac 29040 ctggcagtct gcttataata tttttaaaaa gataaaggac agattattct gggctgtaat 29100 aaaagatgca ttagagaagg caagactggg gtcccaaaaa gcaatgagga agacttgtaa 29160 aggattggta aaggtaagag aatatggtgc ctaagtgaaa gcagtggtta tgaggctaag 29220 gaacagaagg tttaatggaa agaacttttt gaatcctgat acggtttatt ttccttttta 29280 tcatactgct tctcatggag ggagtgggga aaggatttta tgtttagtca ggtatcaatc 29340 cttggtctcc ctatcccaag gaaaagtatc aagttgatct tgtagggtga gcatagtctt 29400 cctactggga cagaatgctt agtgagcaca ccctggctac catctcatct cctctctggg 29460 ttgggttgtc acatttcagt ggccattttc aatctgtttg cattatatca ttccgagctg 29520 cccagagcct tgagttttga ttccagtacc tagtcatttt cttcttcact attttccttg 29580 accttaaaac ttctatttga tcaggcagca gccacccaaa atttccttag gatatagtct 29640 tatccttgac tgtcttagct gaaccttgga gccttcccaa ggtggttttt aactggcctg 29700 ttaaaaacct gacctgttga gagctggttt tgatcatgtt gaagatataa taaagcaaac 29760 aagcttttat aggatatgaa aaaaaccatc agacccacta atttttactg tagaatgtag 29820 gctaatggtt taagtcagtt aatagtacag ttcttcgcca ttttggcatc ttccttcaca 29880 atcctgttcc cattatctcc actttttcaa ttactgagtg ccccatcatt tagatatttc 29940 attccagttt atactactat tggataagac atattcattt agcaatccta tactgagcac 30000 tttctaatca tctggccaac atcattatca tagctaatat ttgagtattt taaaaatgaa 30060 ttttacatag ctttatctca tttaacctca cactaacact aaagtaagta ctaatacagg 30120 ctgtatttca taactgagga aagtgagaga attaatatct tacgcagaac tcatagtttg 30180 taagtgataa aggcgtgatt tgatcctaag ctagttgatt ccaaaatgtt ggtttttaag 30240 tattattcta tcctgcctga taaaacagac agttcaacat aaaaccttaa ttggagagaa 30300 ttataaagta gccagtgata ttgtgttagt aattctaatg aagaagaaaa agaaaaaaga 30360 aaatcaaact aaaaattggt atcagtggaa aagtacataa agtatattcc aacagaggac 30420 gtttgagatc tgtcactaga agaacaattg aggaagttgc aagaagaacg aacttacaaa 30480 gtagttacaa agaagtttcc agtgtattta ttccttattg tcaactggtt atatgctaag 30540 aatatacacc tttaagcaaa tgcccaaatt gcaaggatat aatccagggt actattcata 30600 catttctgta ataaagaaaa cccaaaccgg tggataaatt tatatagaaa cacctttaaa 30660 atattgttga acaattgatg ctatctgaag ttagtaacct taaattagta acattagtaa 30720 tcttaaatta gtaacatttg tgttctacag tgttttggtt ctagtatctt gttttcaaaa 30780 aagtagacag tatcacagat tatatttaat attcttgcct atttgcagta gaaaacttgc 30840 ctgtgatgaa tatttttgta tttaaggcag tggagatggt atattagtat atatgtatga 30900 acatatgatg aataaaagtg ctattcatac aacatttcag gaagtaataa atttggtact 30960 ctttctaaaa gctttgagta ctctatcaga ctatacttta aaagaactga gaaccaagaa 31020 cccttgagtt tgttagagtt ataggtgcaa attttctttg gtgcttttca ctattttgtt 31080 ataaaatagg aatttgtatt ttattgccat tgattaaaaa aaccttttct ctaatttgtg 31140 agaaacatct taataaagca ctttaaaaag aaaaaaaatg tatagattac atcactgttc 31200 tactcacagt cctccagtgg tttcttattc taggtctaaa ctccagagtt tttattaccg 31260 cctctctgac tttacataat ctgattccca gctaactttc tgaccttgtt tctttcctta 31320 cttcccctta ctaactttgt tacacccaca taggtagaat tcctaagggt tgtgggaaca 31380 taccaagcat gtttctgtct cagagtcttt aacttcttcc atctgctagg aatatccttt 31440 tctcagagag ccccgtggct tgctccctta attcattcag gtcactgtta tatgacattt 31500 tattagagag gcttgcctgt gcctatcgta tctaaaatag tatctcccca ccttgtcaac 31560 caatctctgc tcctatatgg agcagacact ttggtgattg ttttttactc tattctcttc 31620 tagaatgtaa gtcatagaac cagggacttt atctgtttta ttctttatgt agccccagag 31680 cctagaatgg agtctggcac acagtgaatg gatggatgga tggatggcta tatgggtaga 31740 tggatgatga attggaatgt ctggcttttg tttgaaacag agatttgtgg ttattagaca 31800 taatgataat ttaattgatt ccttacatct ctctagctct ttaccatttt cagtgatttt 31860 atgctcactt tgcctcattt aatccattca gtggccttct gaggtaggca gagagctgta 31920 gtcattaccc ctgttttata aattaaaaat gtgagactta acttagaccc ttgcccaagg 31980 acacagggct aagaagtaaa aatctgggta ataacctaga cctttctctc tctgacagat 32040 gttaatatca ctgcccaaag ctatcaccta tggaaaacag aatcttttac tgaggaaggt 32100 gttgaaatcc ccttctctac agttttataa agcaaacaaa cctagtgttt acagtataca 32160 tcaggggagc tccagggcta cctgatgatg aagagccttg ctgagtcaac tgtattccca 32220 cattaggttt ttagttaaat tttgttgagt ggaatagtgg ggagaaaata aattagagca 32280 gaaaaatatc gaaatacata ttttgacagg gaaaggacct ctattgagtt ttggaactta 32340 aagcgattta aggtcatctt tcaaattata acactcctct tccttcacat tcctcaggtg 32400 attaaacttc aaaccaagag gacagattta ttgcctatgg tcaccacaac tgcttatata 32460 tgcccggcac tgtcagtttt taacttatgc tttgtgctgg aggccctgtt aaaatctatg 32520 ccagtttttc ttactttagt atgtcaaatg tggtaaaaga cagtctccca cagtcagtac 32580 ctctagtata ttattcctta gttcttccat ttatcccgtt attctgactc tggaccttcc 32640 tcttaggagg ctccaggtgt caggcaggag gggctctcat cctgtctatc tcctatacca 32700 gatggtcact actttaggac atggaccatc tttttcatct ttgtacattt atgtcaacag 32760 tacctatcac agtcttactg agcacaaagt aggtgctctg aaaatacctg aaactgttga 32820 attatatcag tatatattaa aagatcccca ttgcctctcc agttctttgc tgatcaaatg 32880 atagaatcat ttggtatcta tcaaaaatct catattttgg aggcattttg tctcttaatt 32940 ctgtgtaata aacagtaatg gtatgctatt tttgcctgga cctgccctaa aacctgccac 33000 tcttggccca gctctcttat agtttgcatg gggaaatcag gttgtcacag attgtaagca 33060 gatctaatta gtggtaagtt ccattccagg gtagggcaga aacttaggga aatagtatcc 33120 agtctgggga gaaattcatt gttctcttta gataacatga tcctgatgct gatgttcttt 33180 atcttcaggt tgaggtgagg acccctggta atgtaatggg ttcaggagat ttttgcaaag 33240 agaggaaaag ggcagggcaa aagcctgttt agactgccat ttaccatatc aggagagagt 33300 aagaaattat agtgatgaat tttgaaaaat tagaaacttt tgatctcttc cctacatttc 33360 atactttatt tcctgttcag tgtggagaga aaaattcata gaataagaag gtactgtgag 33420 cttgtcttca ggggttgggt tgggggagag gcaggtagaa aagctgtaag ctagcttccc 33480 attcacagct aggctttgag tgggagaagg aagccccaag ggactgagtg gatagtgatg 33540 gctaaaagaa gagggagaaa cagattgagc ttctgagact agctacacaa aggtgagagc 33600 ttggaggcta atgtgtattt cagaagggat atcaggatgg ggtttctcgt gggaggtttg 33660 gtgatcctga tagttaagtt ccatggcagc tggaccagat gcatcttgct tttcttcttt 33720 gcacagaatg tgaaggctgt gtctgagact cctgcagtgc caccagtttc agaagatgag 33780 gatgatgatg atgatgatgc taccccacca ccagtgattg ctccacgccc agagcacaca 33840 aaatctgtga gtctttgggg acctggccag ttttgtgatt tattagagta agtggggaga 33900 gggaggagtt ggtgctgata cctagaagtt taatattctg gtaggcgtcc ttagattttg 33960 gagctggtga aatcactgaa ctaccttgac taaaggtact ttctgtatga ttaaagacaa 34020 aaaatcatgt gtttatttat cattggtttg cagatagtcc caaagtgtcc taggtaattg 34080 atgtttatac cagtccagtc tcggttctcc acagatagga caggaagata tataggattg 34140 atgctactta gtgcaggact aacataaatt attccagtct ccagggaagc aacaaattgt 34200 cttgctacag atcttttctt ctctgttaaa tgtgcagtgt tcagcctctt agagcattat 34260 tgataatatt gaaagaccca agagaatata taaatacatt agtgctatta atatgctggg 34320 catcaggagt gcattttgat gccggatgtt ggcagtgatt tctatttaac ttcctttgag 34380 gttcctgcat ctctgagaat agccatggtc cctctaacct tgctttgaaa catctagtcc 34440 ctgtgccttt tacttaattt ggcatcactg agcactgacc taccatcaga tgatactcta 34500 ctagtgtttc tttgtttagg tcttttacta agatgagagc tccataatga aggaaccatc 34560 tagcacttct gtgatcatga aagaaaaaaa aaagggctac ctctggaata cagcattcag 34620 ggaggtgttc aaacagaaat tggttgaatg actattggag atgctgtgga ggaaattcaa 34680 aggaattata tgacctatta ggatgagaac ttacaaacag aatctgtgga atcatctctg 34740 taggtcacaa agttacctca agggaaaatg cgcttcaaaa ttggagagtc gttaacactg 34800 atatggtttt ttttggtttg tagacatgtt catttaaaac atttttttcc ccactaaacc 34860 atgaacacct caaaaagtag aggaaatcca tatgtatgta tttctagtgc ttaacatagt 34920 tcttggcacc tgcagaggtg ggtaaatatc agaggacagt aaatattgaa ttaattaatt 34980 aattgatctt catagcattt ctttctaaaa aggtattggt aattcctaat ttatagaaga 35040 agcagctgag atttggagag gtgaattgat ctgcccagtc tcttagtaaa caaaagagcc 35100 aggatttgaa agcaggtaat taaccccaac tccaggttgt tttccaccat agcacagtgg 35160 tatttttgtg tgtacaaacc tgtgctagga gccagagaag gagactatta aagaaggaaa 35220 aggcaaggtt tctgtcttca aagagatata tcaggacagg caaaagagga caaggcatgg 35280 aagtacaggt ttaggtgcct gggatatagt ttgtctacag gtacaagaat agaaaattga 35340 gaaatcatta tgctagagta gtaatcagga aggaagccat tgcctctgat ctcagcgtct 35400 tggcttattg ctgttcccac tactggaatg ttctctccca tttccaccta ttaaaagcct 35460 gtccagtcaa aaggtagcat ggtcaagcag tccaaggtgc aggatgtagt tggctttagt 35520 ctctgtggag ccatgggttt gagtcccact gttgccaatg gctttgcatc atcgcagata 35580 aatgatcatc atagataaat gatcctctat ctttgtagaa acctatccgt ccttcaggat 35640 ccagttgcag gatcctgttt gtttatttat tccatcaaat atttacctga atgcttgctt 35700 tcttttagta agtggttaga tggaggaact cacggatgaa tgacccacat tccctgtcct 35760 tatactgttt atggactatg caaaagataa ttgtgatatt tttatgataa aatctgttaa 35820 ataccttgaa gcatctcctg gggaaaggaa gtataattta tgatcacttc tgaacctcag 35880 tagtatatag cttgcttatt tctttataac atgtatcact ttctgccttg tagttatgat 35940 tttctcacct cctttataag aatgagaggt tggggcagca ttttgtacag ggtgaggcaa 36000 caaatagatt ctcagtaaat gtttgttatg taaatgaatg aataggacat ttaatatttt 36060 taaaaggtaa aatttgagtg cttaatgtct gctggggatg agtctgatta cctcacatat 36120 taatcagttt actcctacag caaccccaaa agatagttat cattattatc accattttat 36180 agatgaggaa actgaaacac agagaagtta agtaacctgc ccaaagtcat gcagaaggta 36240 agaataataa gagttgggat ttaagttcag gcagtcaggt tccagagtcc atgttctaaa 36300 tcatcacact ctactacctc ttgatgaaga acaaaagtag ttccttccct gcaagggttc 36360 aggatgtaag gagggagtaa ctcaatagaa gttggtgttg tactagtaat gcaaagtgct 36420 atttgcatta ctacaggata tgtcttttaa cttggggaag cataaaatat tacaaatgaa 36480 ggggattctt acctaataga attgtcttca actaatttct tttgttacag aatatttctt 36540 ttagcttggg gaatacagga tatttctttt agcttgggga agcataaaat attacagatg 36600 aaggggattc ttatctaata gaattgtctt taactaattt cttttgatag ttgtctagcc 36660 tctattgaat atctttagtg ctgagactct cacttctttt gtttacagtg agagtaagga 36720 aggccatttt acccagaatg ggtgggcctg ggcacagagg aggagtaata tagattttga 36780 cataagagaa aggatgagtt tattggggtt tgctctgtgg atgttctaac ctcacatcta 36840 ggagaggttc ccctatgcta acaaaggctg gtcttgccaa aattttgcac taataatgca 36900 ggtatacaca cggtctgtga ttgaaccact tcctgtcact ccaactcggg acgtggctac 36960 atctcccatt tcacctactg aaaataacac cactccacca gatgctttga cccggaatac 37020 tgagaagcag aagaagaagc ctaaaatgtc tgatgaggag atcttggaga aattacgtaa 37080 ggaatttgta gctttctgaa cctttcttcg ttttatgact ggtcaagcac agagatttgc 37140 tggtcgtagg cttggtccat gcagctggct ggccatgctt ggtaccttag gcacagactg 37200 cttctcctca ccctgcccct gaacctccct tccctgtctc acagccctgg ttggatacta 37260 tgttaggatg gagatgtggt gagagtgtgg atagcttact ttgagggcag ggcatccctc 37320 acccctaggc atgtattata gcgaagacaa atagtttttc tctaatgaat agcctagaat 37380 gccatgttga gaaagactga ggacctagtg gaaaagggca ctgtgatcaa ttattgatgt 37440 tttgccattg acctaggaga agacgcatct gccatcttgg ttgtaggaaa agagggaagg 37500 gaagttaaca ttttatgaac ctatagtatg tatcaaatga caagcaaaat attttcttga 37560 ttttttttaa atttaattct cttattaacc ctgtgaagaa aagcattatg atcctaattt 37620 ttgcaatgaa gatatctgag gctcagggag gctaaatatc ctgcccaaaa tcacgcagct 37680 aaaaagtgac atggctgagg ttagaacaca gtttttgttc ctgtaagtag agcatactgc 37740 ctcttgctgt caagtggaaa cttccatttt actttcttta gctgccagaa atgggggtat 37800 gttagggtag cttatttctg tgtttcagta aactgcagaa gaattcatgt tagaaggaag 37860 aactaggttg atctgtgggc ttggaagaat catttgacaa gggctttaat ttattcactg 37920 tatctactta cttccaaaaa ggatttgatg tgaccgattt ggcgacgtct gcaaactggc 37980 attgctttgt ggttctctag ttcttcctga ttaattccta tatgacttta aaacaaattt 38040 tcactgtttt aaaaattgcc cttttctgga tccctcataa aatgacaaag taagaaaagc 38100 cacttaccta gtcatctgtt tactcctgta tgtagtcttg ttctctaaac cagagttttt 38160 tgttttgttt tgttttaaca ataacactta gaattatgac ttagaattaa gacttggcct 38220 gggagttagg acacttgagt tgaagcttct gctcagccac tgacttgtgt cttaggcaat 38280 tttctactgt tctctaaacc tcatttttct catttttaaa atagggatgt taatacttgt 38340 ctcatttgca aatcaaatga ggtaatatat gtgaaaagta ccaattccta gtaattgggt 38400 attaatggag agcacattgg accgggagac taaaaatgaa actcctaatc ttagctctga 38460 caccaatttg ctgtgtgacc ttggggggga agtatcttct tcccttctgt gggcctcaat 38520 ttctccatct ctaaaataca agttataaaa cccagtggaa aacctctatg tcgttgcact 38580 tgaaatattc tttgttgtaa acctcttctc tactttatct ctctggcttt tacttaaagt 38640 ggaaatcagt ccatatatca cctttctgaa gtttttccag tctttttttt gtgtgtgtgt 38700 gcctctttct gtaccccttt ggcttttatc acagcaatta ttatattttc atggaacttt 38760 atgcttactt atctgtctcc tccatgaaat tgaccttctt gagagcaggg actgccttga 38820 ctgtgtcata tctcttcatt cctaacacaa catatggctt ggtgcgtatt aaatgagtaa 38880 atatttactg aatggattaa ttaaaaaatg aatgactttt aggagtttag aaatggaata 38940 gcttactatg aatcaaagca gtcaaggtga tgaaactttt ggttgagact tatgggggaa 39000 aagctgaggg cctgtttagc atgctttttg gctattggag gtagttcttt aacccttgcc 39060 agctacaggg gttggggaaa tgtggaacct tttttgtatc ttgatttggg aaaaaatgat 39120 tcatggtctc tgtttctttc aaaggaagca tagtgagtgt gggcgatcct aagaagaaat 39180 atacacggtt tgagaagatt ggacaagggt tagtaggggc attttctttc cctggagaaa 39240 tgactttaaa gtgtgtgtgc cggcatatat gattttgctt tttttttgcc tctcataaac 39300 cttgttcttc tttctccact ttacccctca cccatctgga gttgttatgt gagactgact 39360 tgagatttgg cagaattatg cttaatatga gagagatgcc attagctgac catgttgcta 39420 aaaatacccc tgcagtgaca gtgagagaac ggtttttttt cttagcacaa aaccaacttc 39480 ccaaattaca actctgcttt tccggatgaa ccctgtttgt cctagtgctg agatccacca 39540 gcactaatat aaaatggaag acccaaaact ataaatcctt ctactactgt gataggacct 39600 gtttttctag gtagatgcca gaatttatcc ttcttgcatt catccttctc tgtatcttat 39660 atgctgccct cagtgtaatg aattcagcaa gtcttaactg agttcctatt gtgtgcaagg 39720 cactatgaat tcagagacaa tctctgcttc caaggacctc aagtcaacag aaaaagatag 39780 gcaaacaatt gtatagtcat atgttgccta acaaagggga tatgttctga gaagtgcatc 39840 cttaggcagt tttgtcatgt gagcgtcata gagtgaacac aaaactaaat agtatagcct 39900 actacacacc taggctatat ggtatagcct cttgctccta ggctacaagc ctgtacagca 39960 tgttactgta ctgaatactg taggcaatta taacacaatg gtggttgtat atctaaacat 40020 agaaaagata cggtaaaaat acagcataaa agataaaaaa aaggtatatt tatgtagggc 40080 acttaacatg gatggagctt gtaggactgg aagttgctct aggtgagtca gtgagtggtg 40140 agtgaatgtc gaggcctagg acattactgt gtacgctact gtagacttta taaaccctgt 40200 acacttagac tgtacattta ttaaaaaaca aagtaattgt gctatgacgt tacaactgct 40260 acagtgttac tgggtgatag gaatttttca gctccatcat aatcttatgg gaccaccatc 40320 atatgcggtc catcgttgac tgaaacgtcg ttatgtgaca catgactgtc ttgtgttttt 40380 aaagtgtttg gctgttattt tgagtgaaag aagctagata tgaaaaagca gatactgtct 40440 catttgattt atatgaaatt caagaacagg caaaagtaat atatgataat agaagtcaga 40500 atagtggtta tttctgggag gaggatgtag agtgggaata ggcatgaagg aatcttcggg 40560 ggtaatgact gtagtgtctt ttgaaagtgg ccattatttt gagtgaaaaa agccagatat 40620 aaaagagcag atactatgtc attctattta tatgaaattc aagaacaagc aaaactaatg 40680 tatgataata gaagtcagaa cagtgtagtt atttctggga ggaggatgtt gagtaggaat 40740 agtcatgagg gaatctttgg gggtgatctt ggtagtgaac acatgggtgt gtacatatat 40800 aaaaatcaag ttgtacactt aagatttgtg caaattaagt tatacctctc tataaaaaaa 40860 aaatgagatt tctaaagacc caccaaaaga gataaaatta aaaagaaact gaaaaagaaa 40920 aatgtctggc tctcagtgtt agtggaatga aagcattgtt gctaatgatg tcattgaaat 40980 tttctttttt tttttaaatt atacacccaa gaatttctga aaataaaaga ttgcataata 41040 tagggaggtt ctgtttatac agcattctac agttcacatc tgttatttgc atttattgtt 41100 taatcctcag aacaactctg ggcttgagtg gatcttattt ttttctagtt ccagtgtttt 41160 tcaaaataga catacttttg tgagtttgaa ctggtgtaag gtatttaaca ccttgagaaa 41220 aaaattattt tgcttacctc agagtaatgt gggatggtat ttcctttagg tagtgaaatt 41280 gagatattaa gaatgtagca gaaatgtaca tggacaagca taggcaagtc aggtttcttc 41340 tctggactgg agtttcttcc tgtaaattta aggaagacag attgtgaatt gaggaccttt 41400 tacctccaga acttttgtga cttttttttt ttttttagtt actgatatat gagacctata 41460 aataggcaac atctgcataa tgttctttcc ttgcttactt gacaggtctg ttagtgctaa 41520 gggaagaatg aaatattaat taagcactta tgctgatgtt tccacattgc ataattttta 41580 aaatataact gtcacaccaa tatgatgaat tggtaatatt gtaatttttg cttgctttta 41640 tttctccttt aagacctgaa gattattatt cttttggact tggaattcat atgtctaatt 41700 atatgtctat tcaagttaga aataggtatt aagcagtggg cctaagtagc tttctacaac 41760 tttgtaatca tggagacagt cccctgaggc ttgggcatgt ttgtccttta agaatattac 41820 atctagtagg atcaggaagg gatggggata ggaagacaga taccagcttt ataacagaag 41880 atagctaggg gcaggataat caggcagtga agaaataact gcctgtacaa gtcatccaat 41940 ccttatttta tagccagtat gttatagtca cagtttctag gcatgtagct agggtcccaa 42000 ctaaagagtg atgattaggg agctggtgac catagtttct agctgcaagg catgcttaca 42060 catgactttt ccccctggat tgacctggaa ttataatctc agggcttgcc taggaagcag 42120 tcttgtaata gagcagaatt ttaattttct tcaaattgca tcatatgtac ctaacatatg 42180 gcatgtacag gtacaaatat gtgttacaaa ttacttacct acaaatacac agggtttatg 42240 gtcttaagca taaaatgagg gttttagtta ttttgaatgg atttagttat tttgaatgaa 42300 ttattacttt cctagcatta tttctgcatt tcttcccccc ttctctgtct gtatatatga 42360 atgtgtatat acttaacata catatgtgta cacacatgtg tcaagtgggg tcattcttac 42420 cttgggggtg gggcagaggt accctcagat cttctgggag catgtatgtt tgaaagagta 42480 tatttaatat cattttatgt ttgaaaaatg aaaactaaaa accactttta taatacaaac 42540 tacagaaaac aaaattcaca tatcctgttg atggaataaa tgattgaaaa ccattatttt 42600 attttaaagg atccaagtgt atcctgtaat atcagagtgg gaagggtact ttgagattag 42660 attattcagt gtggttcttc aagtttttat agttttttac tatttatagt tttttaaagt 42720 aaattttcac taaagatagc atacataagt gtacaactca atgaattttc aaaaagtgaa 42780 cacgcctatg taaccagtac ccagattaag aaaaagaaca ttaccgacac cctggaagtc 42840 catattcctt tttgataaat aaataagata tgctggccca cccccaaaca cattctgaaa 42900 ttctccgcag ggcccctttc tccccacctg tcaccccccc agttgaaaat catagatcta 42960 atcttatccc ttttatagat gaagagtttg aagccacaag aaattagagc cacgtgccaa 43020 gggtgatgga ggagaggaga gaagcaagca ggaacaggaa attaatatat attgagcatc 43080 tgttatatga tatgcagttg gctaagtact ttacatgaac taatcaattt aaattatctt 43140 gtattctctt atgtccactt tacaaatgag gaaagtgaag ctcaaagaag ttaaataact 43200 ttcttaagat cctacctagt aggtactagg tagaagtacc aggttggaat tcaggtctcc 43260 tggccactag agcctacatt ctctgtacta cctcttatag cttcttaaat gtgagaatgg 43320 atgctgcagg gagaggatta tttttactgc tcttgttttt gacactcaca gattgtgtgt 43380 agaactgcat ttgacaaata tcactttatt gaacactgtg tttttttccc tctttctccg 43440 ccaccttact gttcagtgct tcaggcaccg tgtacacagc aatggatgtg gccacaggac 43500 aggaggtgag tatccaccac ccactgttgc tcctttagtt ctgtttcttt ttatttggga 43560 ttaattttta gcccttgtta ggtcaacagg aacagttcta ggtttttttt tttttttttg 43620 ggatggggtc tcactctgtc atccaggctg gagtgcagtg gcgtgatctc tgctcactgc 43680 agcctccgcc tactaggttc aagcagttct ccagcctcag cctcctgagt agctgggatt 43740 ataggaacct gccaccatgc ctggctaatt tttgtatttt ttagtagagg tgaggtttca 43800 ccatgttagc caggctgctc tcgaactcct gaccttaggt gatcttcctg cctcggcctc 43860 ccaaagggct aggattacag gcatgagcca gtgtgcccag ctggaacata tctagtttct 43920 tactgtagca tacaaaagcc aaatagcagg ataacattat gaaacttaag gtttggacta 43980 aggtgaaata gttagatgga gggaagtgga tggatttgag agataactta gcaggtaggt 44040 tcaacaggat tagatgattg aatgtgaaca ttacttacaa gtgggtgata ttattcaagg 44100 agaaaggaaa aagaaattgg atactgcctg attgccaagt ggatagttgc ttacataact 44160 ttggggcttg ggggagaaat ctgatctaca ggagatttaa accatgtctc ttgactccta 44220 atctactggt gctaccttta ctttccactg aacagtgaaa tcctcattta tttggaggca 44280 gctttttgtc cctctcattt cattaagcca tgagaactat agctaaagcc tgctgaccca 44340 aggtcttata ttcagtagaa gaaagatatg aagggaatat agagtacttg aggtttcctt 44400 gcattttaat atttggttgc agagtaggag tgtcagccaa tgaactgtgg atggcagctg 44460 ccccagagaa gttcttactt caaagagtac cagtacacaa taattgagag aggcacctcc 44520 tcttccaatg aaaatagagt tgggcttctg tgagggaggc tgctagaaac gtgcttctat 44580 atttttggtg gttgaaacca cgtggcaccc tggagaagtc atggttaata ggggtagcac 44640 atatctcact aggctcttgt gagtttttac aagtgttaaa gccaaatatt aaggctccac 44700 ctagggtaag ccaagtttta aaaatcatta ttatggttaa ggatttattc attattcttt 44760 gatgtgagaa tccaaagacc tagatgctta taagccttta gctacttata taaccttggg 44820 caagtcattt gacctctctg agcctgaatt ttatatattt ataaaatgtg cataataaaa 44880 tgtccacgaa agtgttgaaa gaattcagta aagcaataca tgtgaaaatg cattataaac 44940 tttatgaact acatattcat atttattcat ttttactcat atacatgtgt atgtattcca 45000 caaagcattt gctttatgcc acaccttatg ctaagaattc tgtgaaagtt cacagacatt 45060 aaaatcatat gtaaatgtaa gagaccataa tttcctttgt gtacatataa aagccttgta 45120 atattgacac gaatctgcct taaagtaaaa ataatggtga ggaaggaagg ataatattat 45180 catggcattt taatgatggt gaatatgatg cagagattaa aataaagaag gctataccaa 45240 tcctgaaaag aactctatga ggtaggtaat attatttctt ctttactgct gaggaatttg 45300 aggctcaggg tgtatagata actttttttt gcaaagttgt actgtcagca attaccaggg 45360 actggattag aatctttgac tcctaatcct gtgtgttttg ggccgcaaga gcacaacaga 45420 gctgaggtta gatactagag tcccttctgg cctctcaggc ctcacagtcc ttgatgggag 45480 gcaggtaggt gatgatggtg gttcctggaa gccctggagg ctggctgtgt gagaagctgg 45540 ccagaggaaa gagaaattta aattcattgt atgcgtgtct tgtggttaca aaactatgaa 45600 tggaagggag tccatcaggg tggctaatgc agtgtggtca ggaaggcttc atttcctgcc 45660 ccagggagaa taaatcttta ttggagtcca tgggatgctc tcagtctttg tgagttcttg 45720 cttattgggg gttatccagc tgagaagggc agacctagtg tctcacatat aggaagaatg 45780 ttagtgctgg gaagtcttcc agttaacata tccaacaatt atttttcaag tgtgttttta 45840 gagagaatgt gtacaataga aagaacagac tggaagaggc tttggagtca catggacctg 45900 taggcattga ttatgacctt agaattttct tatgagtctt agcctcaatt ttcttactga 45960 aaatgcagat catgattctt actttgtagg gttgtgtgtg gttaggtttt actgtgataa 46020 tataagtacg tcatctagca cagagcctgg cacacagttg gtatttgata aacagtagcg 46080 gtgggagagt agttgctgtg ccaagtactt attaaatgct gttattactg tgattaccat 46140 ttttgttatt gtctaaattt attgcttaaa ttatttactt atatgtgagt aatgcattgg 46200 ccattgtctc acttggtatt caaaacaatc ctgaaaagag gggaatcaag gttgagatta 46260 tcttcaatgt attgatgaag agactgaggc tcagagagct tgtgattggt caaggtcagt 46320 aagtgacagt cagatcctga gcttaagtcc tcagagttca agtccagttc tttttctgtt 46380 acaggaatgc tatctcctag cctatatagg tggggcttag gtgaaggaca cttagcctag 46440 cccatagtta gtagctattg aaaaggactc ctggccaggc gcggttgctc atgcctgtaa 46500 tcccagcact ttgggaggct gaggcaggca gattacgaga tcaggagttc gagaccagcc 46560 tggccaacgt ggtgaaaccc tgtttctact aaaaatacca aaattagctg ggtgcgatgg 46620 cagatgcctg taatcccagc tacacgggag gctgaggcag gagaatcact tgaaccctgg 46680 gggcagaggt tgcagtgagc agagattgcc attgcactcc agcctgggca acagagcaag 46740 actctgtatt aaaagaaaaa gtaaaaagaa aaggactccc atgtccttcc ctagcctcca 46800 tgttagcctc tttctgcctc tcctcccctg ctgctgccag cagtgaccac tgtgtggttc 46860 gtaagcagtg tcccatcctc caagatggca tgagtaactc aaaagacatt cttctggctc 46920 cagtccttaa tgtgcgttat aagggctaac tatacttaat atctttagat caacatgttg 46980 cttttcaaac gttaggaaaa actaatgctc agctgttata gacagctgca gccagaccag 47040 aagcctgacc taacaggaaa ccccactcca aagcaatttt tccttacttg aggtagggaa 47100 tggaggtggt cagatttttt tacagaatta cttctattct aaatgattaa cctggtctct 47160 gcaagagact gtccattctt gacttgaaga aataatagag tcagggattt gtcacataaa 47220 agtttgaatc attccttctt tatacttctt gctgcttcta gaaagacaac ttagttctgt 47280 tagatgttcg tgttcttgtc cactaagcag gaaaaaggaa aaatatctct agagaatgaa 47340 cacttagaaa ataatttaaa gtatacttct atgggataga ggagcccatt gccatttttt 47400 ttttgttttt atttaaaaac aaagtacttt ccaacctact gatttctctc ttgccatctt 47460 cctttctaca gacatttgtt gagcaccaac tctgtgtgta aaggcctgtg tttggcattg 47520 agggttctgg gctttcaatc aagccctcaa acctctcaat acacacaaac atacacacta 47580 atcattatac atgatagcaa gttgtaagtt ccataagaga gggataaata tgatcttata 47640 gggctttaaa gaagggaaaa ggcatattgg gtttggagaa tgaagtttcc tttggcctga 47700 tacaaaaaga gtaagtagct tttgaatgta tggaaatgat acaggctgtt ccagatgaag 47760 agaagattct gaacaaagga agggggacta gataccaggg agaacatgga gtgttctcca 47820 tgaacactct cctctgtgaa ggaatagagt tggaagtaaa gtaggggcca gatcatgaga 47880 actttgaatg ttgtactgag aagcatttct aggcaaccag gagccatgta agtttttgaa 47940 caaggttatc aaatgtgtac tgtgggggaa aattaatctg gctacatgta caggataact 48000 tgacatccct tttgagggtt ctaatcctga ttttgctatt gaatagctat gtatgcaagt 48060 cacatcctct ctgaaacaca gtttccccgt ctgttgaatc attagtaata tttagcacac 48120 aaggtgtctt tgaagattaa atgaaataac aaatttgaga acaccttata atagtacctt 48180 acacatagaa ggaattgata ccaagtacct tagaaactaa cgtataccat atattcatgt 48240 tgtctggaaa tttgttttta tttctgtaga tagagctctc atcctccttt tctcttcttt 48300 cttaacctga tcaactctga tttatctttc tacgccaggt tcagaaagtc acttccccct 48360 ttatgaagcc tctataccta gccagattga aagcattctt ggcttttgcc gtattttatg 48420 tttcgctagc attttgtatg agcctctgtt atagcatctc ttttctgttg tgacttttta 48480 aaagtatttt tctacaccaa agtttgattc tgaaactttg atgtgtatca cagtgacctg 48540 gaggactttt tacatcacag agcttctgat ttagtagatt tggggtggga cccgaggttt 48600 ttatttccaa caagctccca gatgatgatg atgctggctt gagacctcac tttgaagacc 48660 actggtctac atagacaaac tgtgaattac tcgagggcta agactgtatc ttactcatct 48720 ttgtttcctg agtacttaag gtctggtact tagcaggtgc tcaataaatg ggaatgacta 48780 tggttgcaca ttgaatatac tcttgatttc aggtggccat taagcagatg aatcttcagc 48840 agcagcccaa gaaagagctg attattaatg agatcctggt catgagggaa aacaagaacc 48900 caaacattgt gaattacttg gacaggtatg gagtggtggg tcccatcaga gggaaagggc 48960 ctttgggact gattggtggt gaagctgtta gagaagaggg cagtgccagc cctcagaact 49020 gcctccattt atgagtcttc tgtgatgctg agctctctgt atgtgaccct gtgtaatgct 49080 ggacaccttt tttgctggga aatccttctg tgttaggctg aactctacca ccgtgtagct 49140 tctaacagtt gatcctcgta ctgtttgttt tgcctaaggg aaacctacca acccatctgc 49200 ctgagtcaaa catctaggtc cttaactcat tttacatccc aaatctttcc ttattctgct 49260 catcctcctg aataacttgt tgtttgttta tgttctctta aaatgcagtg tgcagaacta 49320 gatgtttcat ttgagatctg accactatgg agagagtggg actctttcta aactcaacac 49380 agcttaatga tttcattctt gttttatctt ttaggttgtc tgctttgact cccagtaagt 49440 ttgcagtcaa ctaaaatttc tgattttttt tttttttttt acgtaagctt gcattaaact 49500 ggaaatgtgg aactttttca ctatcttcat taagtcccat ttttattatt ttagcccatc 49560 attccagccc atcaaaattt ttttgcaaaa tagtaatagt ggttgtatat aatatttgta 49620 ttgtgcttta ctttgtacca agcactttca tgaacagtat ctcattgtga ggtagggttt 49680 ttttatccat attttacaga caaggaaact gaggtttata gacgttaata acttgtccaa 49740 gagtagtaag ccataaaaca gcaaagcagt gttttgaatt tgaatattcc tagcaccaat 49800 atctagcacc tttctatttt atcatgttct tttctccgat acctgaaaaa aaaaaaaagt 49860 taagcttctt gcccagggtt cagagacatc gtaggtggag gtgaaattgg actaaatctt 49920 ggactctaat ttctttacct cttatattgc atgaccagct acctcttcct gttactggta 49980 gatttcataa gggcgccttc ttgttctgca tctaaaacac tagtaagctt cagtttcctt 50040 aaagaagaaa agagttgaac tttgacattc ccaacactat aaagaataaa gccttcttct 50100 ttgagatggc tgtgtctcct taatgtgggt cctgactggc tctgaagaat cctctgaaag 50160 ttagtacctt atcatctctt tcagaactta ttatacagag aatatagctc atgcaaacta 50220 ctgaaggcga caggggtatt agattttggt tcaggatatg gaaaaatgaa agctgtcagt 50280 aagggtatgg cctgtctcac aaaatggtga gttccccatc agtggatgca tctacatgat 50340 aaaacgatgt tattgaagga atttgtgttc tgggagtagg attaagagac ttatagattt 50400 gtttattaat catatcttta ccagtattcc cagagacttt tactctggaa attagaagta 50460 aataatgcta tctttgccct caaatagttc ctggtctggt atagaaaaaa aaaaaatacc 50520 tctgtaaggt catggaagag gacattaacc tgagcattca gtgaaggtgt tgcaaatgag 50580 ggtctgtggt ttttgtgaga tgtcccagag gggaactcca tcacatatgg atgaactcat 50640 gggcagaacc ctcatgatgt aatcaccttc caaaggcccc acctcctaat aacattagtg 50700 attagatttt caacatataa attttgaggg acacaaacat tcagaccata gcagttcaaa 50760 tccccatagt cttctctctc aaaagagatt gttgaagaaa cacttagtct gtcatgttta 50820 ttagtcagga cattaaaaaa ttataaatca gaacacagag ttttatggaa agagctgtcg 50880 attagtagtc aaaaaaatag gattcaaatc ttacccttgc ttgattgcat ttgttaaaat 50940 cttccaacag tctccacatc tgtccagcag tattcccaat atcaggatga agataagatg 51000 ataccacaca gttcaagcca atgcaagaaa tgtttttttc caatccagct tctttgtcca 51060 agactggcag aagactttag ggctctaggc tcgccttgtt tttatttgtc tgattctata 51120 gtaaggaaac attgaccaaa taggctaagt gaccttttta tttatcgtgt aatagggttg 51180 agtgatccaa ctcctgagtg aaactatcac ttctaactca tgtcagccca taatctctct 51240 ggggatatct tactaaatgg tagtcttggc ttatcagaag gctttggctc cataagccct 51300 tgctaaattc attccattct tgaaccacag acactcaaag tagaggtcaa ttgtcagcat 51360 aatgactatg tatacaacac tttgcagctt ttaaggggct cctataaaca ttatttcgtt 51420 taatatttat agttatcttt tattttttca gtgatttatt tagtaaatat tggcatctgc 51480 tgtgctggac tctgaggata gagtcttgaa caagacccaa tacttggtct catagatctc 51540 agatctggta ggactctctg ggaggtttag tagctctctc ttctttgggt aacatggact 51600 ctaaatccta tctttgtcaa atgtttggat gcttctagag gtgtgggaat catgtcagtc 51660 tttctaattt taggatgtct tcctttgtgt tgtgatgaaa tataatttta ttgtatcact 51720 tcaagctaga gatcttagtt ctgccctttg gggcttcaca gaacatgtat acttggtttt 51780 ctccaggagc atccttgaga gattcaaagt tagtgattct gttccccaga ttctgctttt 51840 ccccaaagta aagaaccttt tactttactt tgtgctaaga cgtagtttca ctctgtgtat 51900 atgcttagcc tttgaaaccc agtgctatgg ctcagtgtac tctgattgtt ctctctcacc 51960 ctttatctgt attcctgcag ttacctcgtg ggagatgagc tgtgggttgt tatggaatac 52020 ttggctggag gctccttgac agatgtggtg acagaaactt gcatggatga aggccaaatt 52080 gcagctgtgt gccgtgaggt aaggccaagg gccaagcagt cttgggtaga aagctgcctc 52140 catattcctg tctcctgaag ttccctgtac agtgagatct cagcctgggc tctgtggaat 52200 tggtagaaat tagtgctgac actgctatca ttcctgtatt gtacctgtgt ttgtcatttc 52260 tatcaccatg attttttttt taacgataac tttattctac ccattgtaga aaaattataa 52320 agtactagaa agtatatatt gtaaaaagtc actcataatt ccagtcttag agataaccac 52380 ttgcctaagc acgttataat atattccttt atctctttta tctgggcata tatttcaatc 52440 atttttattc tgctttcttg ctttatttat gtgctataag gaaaataaaa cagagtaaga 52500 aggtagtgat ggaggaagta ctattttaga caagacagtc agggaaggtt ttgctataga 52560 tttgagtaaa catctgaatg atatataaaa gcaagccagc aaaaatctag ggtaaaatac 52620 ttccaggaaa ataaaaaaag aaaacaaagc tcaaatagat actccctttc tcctcattgc 52680 tattaaatgt ctagaattga taatacgctg atgtaggagg aagaaaagcc ctccagaaag 52740 cctggacagt gtcctcaggg aataaccaca tttcatttcc attagagcaa aaagatgaag 52800 ggaatattca gagacattga ggatattggg tgttttgctg atgacagact ggactttctg 52860 atggcccaat aatgcaaaag taattgcggt ttttgccatt acttaaaaaa aaaaaaatac 52920 caaaaccaca attgcttttg caccaaccta gtagaaaagt taggaaatta ggggaagcat 52980 agaagatgag atgaggttca gaattttgtg gagatgagta taatagtatg atgggggttt 53040 gatggtgcaa gagctgaagg catactgctg aaattatgta gttatatatt taatcagtgt 53100 taagaaaaat aaaatgtgaa atcaatactg ccctagaaaa taatggtccc ctttgcaaac 53160 aactttggga tctagaatgt tcacaagtaa ttggaattaa ttaatggaat taataccagg 53220 ctgggattct taggggttct gacttttttt tttgttccac tactaatgca gtatttcgta 53280 accttctttt cattatctcc ttcttaagga ggctttttcc taatcatact ccctgccatg 53340 aaattgtaat atcacagata tatagctaag ctgtttgggt cataaaccat tgtaatacct 53400 aagatttttt tcaccccacc cccttaccct caggaacgaa gtttcactag ttgagaatac 53460 atgcatatgt atactttggg aaatcatttt gcctcattat taatctgtaa aatggaaatt 53520 gcaacattca cactatatat accttagggg tgctatatgg atcaaaaagc acttggaaaa 53580 aaatgttaat gttgtataat agatggtatt actattcttc agttctaata gagatcatct 53640 tctcctttta aaaaatcttt attgagatat aattcaccca tttagagtat ataattcact 53700 agtttttagt atattcatag agttgtacag ccaccatcat aatgaatttt agatcctttt 53760 tatcacccca aagaggaaat taatatccat taacagtcac tccccatgtc ccctgccctg 53820 actctcccag ccctatgtaa ccactaatct attttctacc tctgtagttt ccctgtactg 53880 ggcattttat ataaatggaa tcatgtaata tttgctcttt tgtaattggc ttcttttact 53940 tagctagcat tatgtttcaa agattcatcc attttgtagc atgtatcagt acctcattcc 54000 tttttattgc caagtaatag aatggacatt tgggttgttt ctgttttcgg ctgttataaa 54060 aaataaaaaa ttactctatg aacacttata taccagtttt tgtgtggagg tatgttctca 54120 ttctcttgga tatataacta caggtggatt gctggtcata tggcaactct atactcaact 54180 tttgaggaac tgccaaactg tttccgtagt agctgtactg ttttacgttc ctgccagcaa 54240 tgtatgaggg ttccagtttc tcaagataat tctttttcat cataatttct gacatcctga 54300 gacctgtttc tccacccttc taagaagagc agctcttaca ttttcaggga aaatccaatt 54360 ctggatattg ttttattctt actttagcca ttggtatatt cttagaaagt aaagtattag 54420 gttaggacca tttaaatagt attttcctgt tcacttatat ctttagtgtt ttttaattgg 54480 tagaagtttc taaaaaatta gttttgggtc aatgaaatca aattattgat ttagtagaaa 54540 agctgagatg atctcatctg attcccaaat tttatagatg gaagtccaga attatatagt 54600 caatggggta gcagcagaac caggcatttt aattgtacca ttgtgtctct ttattactca 54660 ttctgcctgc taagtatagg tgttttccaa gattcttctc tttctccttt accctttgcc 54720 gggttcaacc atttttataa cttaaacaaa aatcatctct tcccaaatga cttgtaaata 54780 tttaccataa tttatcataa attaagttgc ctaccagaca tttgtcttca gatatcaaat 54840 tcagcatatc caaaatagta caaaatatct cctcaccaaa ataagattat cctgctaact 54900 tactgcttct agtatcactt ccatatgtct gcttcctcaa atatgaccct tatttgttgt 54960 tttcctctgg tctgtcatta taagatcctt tccatttttc ttcagtaatt tctcttgggc 55020 cttaggtcct ttaccaagga tctaattaga aaaaaaatta cattggagga actcctttac 55080 ttaaaaaaac tgttttaagg tgaaaggcag catggagtgt taaaagaatg tttagctagg 55140 agatggaaga cttgtttcta gatctggctc tgcgattgat ttggggcaag tccttttact 55200 cactggggcc ttacttcctt tattttaaag tgaagaggtt gaattagtgg attttctatt 55260 aagtcttctg tattatatgt atgtcctttc tttctccccc tgcccacacc acttcaatag 55320 tgtctgcagg ctctggagtt cttgcattcg aaccaggtca ttcacagaga catcaagagt 55380 gacaatattc tgttgggaat ggatggctct gtcaagctaa gtaagtagga gccttaggaa 55440 tgataatact tctctgcctg tggggccctg tctcctgtga gatggtcatc atataatagt 55500 aactacgaca ctcaccaaag ggtctttatg atttcatggg atatgagggc tgttttatat 55560 tttctgtatt aatcatagga tatcatttca ttattttttc attcatttat acatttaaca 55620 acaaccacaa aaaactttgc tgaatggtta ctctgtgcta ggtcctgtga tgggcactga 55680 gagtaaattt tccctacctt ctcaatctag tatggaaaac gtgtaataga taattagaaa 55740 aaagagtttt gtataatttc tttcattcag ttattcagca aacttttatt gaatgtccag 55800 tacatgctgg tccttctagg gatatgagga caagtaagag gacataatcc atactctcaa 55860 aggcttccta atttaaataa atttataatt gcaatataat gtaaaaacca tggcaggtag 55920 agcattttgt tcaaaggtgt aaagaccagt agaccagaca aacaggctgt gtcatgggtg 55980 gatgggcagt ttatggaagg actgctagca gccctgggtc tggggcataa aaacagggcc 56040 atgcctagtc ataggtgatt ggggtttaaa tcaaggactc gtgcccaagg aataagacaa 56100 ggacctagtc gccagatttg ggattatggg aatggttttt caagaacaaa gcagaattgg 56160 gatgagcaat caagaccaaa atgggcagct tggactttaa cactaagtcc cctggtttga 56220 ggagaagtag gaggccagag atggtgtttc ttatattcct tctatttctc ttcagggttc 56280 aataggaaat aggctaggag ctagacatga aggtggtagt caagtgactt tggagagaca 56340 aaggatagca gagctggaag ccaggcggat cttgtcacct atgtgtaggt acacagtcaa 56400 cacttactga agtgagtgta tatcctaaat gtatgttctt tctcttattt ttcttttcta 56460 gctgactttg gattctgtgc acagataacc ccagagcaga gcaaacggag caccatggta 56520 ggaaccccat actggatggc accagaggtt gtgacacgaa aggcctatgg gcccaaggtt 56580 gacatctggt ccctgggcat catggccatc gaaatgattg aaggggagcc tccatacctc 56640 aatgaaaacc ctctgagagt gagtgttacc agttctattt caagttattt gggctgttgt 56700 ctctagtccc aggaaataag aatccagggt gttctctgtg gttttcaacc tggagatccc 56760 ttttctggat acaactagtc actgttgaac ttaaagtata aatggccaaa gaaaaggaat 56820 attagaagtc actcattcat ttattcaaca attatttgag tccctgtcaa gtgcaagaaa 56880 ctattctaat gaaaataaaa taaattaata tgtagtaaca gatggtaaat gctgtgagaa 56940 aaagcaaagt aagggagtta acagagtaat ggaggaacag gtgtgctatt ttagagtaga 57000 gagggaagtt ctctcttata agttatttta ttctgcagag ttgggaagtc attagagggt 57060 tttgagctga ggagtaatgt gatctgattt atgttttaaa cagatcattt tggccgcttt 57120 tatggaaaat agactgcgag agactgttgt aatagttcag agtggtggtg tcttggtagg 57180 agtggaagtg agaagtggtc agattcagga aattaaagga ctagctgaca agatgaactg 57240 aatattagag aaagaggaat cagcaatgac tacaagattt ctggtctgaa ttgctgaaaa 57300 aattcagttg ccatctgttg aaatggtgaa gagcatactt aagttgggta ataaagagtt 57360 ggttttgaat gtgctgattt gaagtgttaa ttggacattt aagtggagct ggagtagagt 57420 ttggcttaag agtctggagt tcaagggtga gaagtcagga ctataaacat aaatttatta 57480 gtagataata tatagttggt atatatagta ggtagatgag atcactcacc tagggaatga 57540 gtacagttaa agaaaagaga tttgagtact gagctctagg ttattccact gtttacaggt 57600 tggaagaact agcaaagtag accaaggagt ggcctttcta gtgtgagaga acagagaagt 57660 gtcttggagg tgaagtgtcc tggagatgaa gagaacagtg tgcttcccga aggaggagga 57720 gggtagatta tgtgtcaaat gtgattgata ggttaagatg agactgagaa atgaacattg 57780 gatttgccaa tgtagaggtt cttaatgacc ttaataatca gttatcagta gagagatggg 57840 gatgaaagtc taattaaatt catgaaagtg gaagaagagg acattgagat ggcatgtata 57900 aacaggtttt tttttctctt aaaggaattt tgctgggaag ggtaacaaaa atggagcact 57960 atcttaaaat ggagtcaagg gagggttttt ctaaaagatg atgtctgtgt gctaatggga 58020 atgatttagt ggagaggagg aaaatggtca tgtacaagaa ggaagtgaca accatagaag 58080 tgatatcctt aagtcgatga gaggggttcg atcaagtact gaagtaaagg acattagatt 58140 agcatggaca atttatccct tgtaacagag catctcaaca caggtgcaaa taggttggta 58200 atcttggtgt gggcaaatgt gaaatttatt ctgattgctt ttattttctc atttattccc 58260 acctcccatc cagtgcagga attccctgtg cactgttgct ggtaggtggt catctacatg 58320 ctcatgtcat ccttgaatat ctctgattat tggaaattgc ttcagtgcac caaactgaaa 58380 ctaccttctg tagtttctat cctttgatcc cagctttgcc tctgactcca catacaacat 58440 actcttccct cccttagaac ggccttgcag atatttgaag atgataattg ctgggatatg 58500 ttggaaataa gacagaatga tcctagacat aggcatattt ttttattttt ttatttttat 58560 tttatttttt tttttgagat ggagtctcac tctgtcacct aggctggagt gcaatggtgt 58620 gatctcagct caccgcaacc tccacctccc aggttcaagc gattctcctg cctcagcctc 58680 ctgagtagct gggattacag gcacctgcca ccatgcccag ctaagttttg tatttttagt 58740 agagaccaca tttcgccata ttggtcaggc tgttctcgat ggtctcgaac tcctgacctc 58800 aggtgatcca cccacctcag cctcccaaag tgctgggatt acaggagtga gccaccacgc 58860 ccggccctag acatatcttt ttatgtttcc ggaccctatc tgtgaagtga agatgataag 58920 tcttgcccta ttacccagag ttaattgtgg ccattaaatt agacagtgct tatgtaagta 58980 ctctttgaat taagtgctga gtgcttgtaa agatttagtc ttactgtttt tctttacctt 59040 tcccaggaga cgcttcattt aggggactag ggtggcaaca gttttcactt acatttaata 59100 ttcctttcct gctagaagag atcagttctt ggatgactga ggtgaaggca ccaggaataa 59160 tttctgccag cagctgggca gatgtttaac tcaacaggct aaatggataa taaacccttc 59220 agatgtgtaa gcaagaataa taataaagcc aggcacagtg actcacgcct gtaatcccag 59280 cactttggga ggttgaggca ggaggatcac ttgagtctag gagtttgaga ccagccaggc 59340 caacatggta aaaccccgtc tctactgaaa atacgaaaag cagtcgggtg tagtggctgg 59400 tgcctgtaat cccagctact tgggaggctg agtcaggaga attgcttgaa cctgggaggt 59460 ggaggttgca gtgagccaag atcgtgccac tgcactccag cctgggcgac agagcaagac 59520 tccatctcaa aaaaaaaaaa aaaaaaaaag aagaagaaga ataaaccctt aggtctgtaa 59580 catatttcat aagtttacaa agtactttca aatacactat cttattttat ttttacagac 59640 atgtgagatg ggcattatca tcatccctac tttacagaca gggaagttgt acatagtccc 59700 atggccttta aatgttcaac ctgatcctta ggtcatactc tttccagtat atatacacag 59760 ctgctacttt gcatcctaga gcataaagca gctgaccata tttctagaac ttgtatatgg 59820 ggaacaagag agagcctcat ttggagctca aggtgattca ccatttttat tgccaggcct 59880 tgtacctcat tgccaccaat gggaccccag aacttcagaa cccagagaag ctgtcagcta 59940 tcttccggga ctttctgaac cgctgtctcg agatggatgt ggagaagaga ggttcagcta 60000 aagagctgct acaggtaact gtccttatgc agggaagcac ctaattcagg gaatcactaa 60060 accaggcctt ttttacttca tgtttgtaag gtactggata ttatagaact aggcttctct 60120 tctaccactt cctacactca ctctgttcct actgcccttt tatccctggg cctttgctca 60180 tttattttct tagcctttct cagcctgtta aaactctaca cgttctccta ggctcagttt 60240 acaatctatt ttctgagcac ctattgagct ttgcttgtag cttttttgct tgcttccttc 60300 cttcctttcc tttcttcttc ctttcccttt ccttcccttc ctttcccttc ccttcccttc 60360 ccttcccttc ccttcctttc ccttcccttc cctttccccc ccctcttccc tctccccctt 60420 cctctcacgc cccacagggt ctcactctgt cacccaggca ggagtgtgca atggcgtgat 60480 tttgtctcac tgcagtcttc ctctcctggg ttcactcaag tgatcttccc acctcagcct 60540 cccaggtagc tcagactaca ggtgcacgcc accatacctg gctagttttt taaaaacttt 60600 tttgtagaga cggggttttg ctatgttggc cgtgcatggt ctcaaattcc tgggttcaag 60660 cgatctgcgc acttcgcttt cccaaagtgc tgggactaca ggcatgggcc accatgacca 60720 gcctgcttgt atctttcatt agcacttcct ttgttctgtt tgaattagtc tggcagagtt 60780 tgtttgagtc ttgttgagaa acgggtctct gacacccaaa tcttacacaa cttatatttt 60840 gctgggatta gctcaaagga tacattttta gcaaaggaac ataagtttat tttattttat 60900 ttatgtttta tttttctttc ttttttttta attctttttt ttttacattt tattattatt 60960 atacttttaa gttttagggt acatgtgcac aacgtgcagg tttgttacat atgtatacat 61020 gtaccatgtt ggtgtgctgt acccattaac tcgtcattta gcattaggta tatctcttaa 61080 tgctatccct ccaccctccc ctgaccccac aacagtcccg ggtgtgtgat gttccccttc 61140 ctgtgtccat gtgttctcat tgttcatttc ctacctatga gtgagaacat gcagtgtttg 61200 gttttttgtc cttgcaatag tttgctgaga atgatggttt ccagtttcat ccatgtccct 61260 acaaaggaca tgaactcatc attttttatg gctgcatagt attccatggt gtatatgtgc 61320 cacattttct taatccagtc tatcgttgtt ggacatttag gttggttcca agtctttgct 61380 gttgtgaata gtgccactat aaacatacgt gtgcatgtgt ctttacagca gcatgattta 61440 taatcctttg ggtatatacc cagtaatggg atggctgggt caaatggtat ttctagttct 61500 agatccctga ggaattgcca cactgacttc cacaatggtt gaactagttt acactcccac 61560 caacagtgta aaagtgttcg tatttctcca catcctctcc agcacgtgtt gtttcctgac 61620 tttttaatga tcgccattct aactggtgtg agatggtatc tcattgtggt tttgatttgc 61680 atttccctga tggccagtga tgatgagcat tttttatgtg ttttttggct atgtaaatgt 61740 cttcttttga gaagtgtctg ttcatatcct tcgcccactt tttgatgggg ttgtttgttt 61800 ttttcttgta aatttgtttg agttcgttgt agattctgga tattagccct ttgtcagatg 61860 agtaggttgc aaaaattttc tcccatcctg taggttgcct gttcactctg atggtggttt 61920 cttttgctgt gcagaagctc tttagtttaa ttagatccca tttgtcaatt ttggcttttg 61980 ttgccattgc ttttggtgtt ttagacatga agtccttgcc catgcctatg tcctgaatgg 62040 tattgcctag gttttcttct agggttttta tcattttagg tctaacattt aagtctctaa 62100 tccatcttga attaattttt gtataaggtg taaggaaggg atccagtttc agctttctac 62160 atatggctag ccagttttcc cagcaccatt tattaaatag ggaatccttt ccccattgct 62220 tgtttttgtc acgtttgtca aagatcagat agttgtagat atgcggcgtt atttctgagg 62280 gctctgttct gctccattgg tctatatctc tgttttggta ccagtaccat gctgttttgg 62340 ttactgtagc cttgtagtat agtttgaagt caggtagtgt gatgcctcca gctttgttct 62400 ttgggcttag gattgactcg gcgatgcggg ctcttttttg gttccatatg aactttaaag 62460 tagttttttc caattctgtg aagaaagtca ttggtagctt gatggggatg gcattgaatc 62520 tataaattac cttgggcagt atggccattt tcacgatatt gattcttcct acccacgagt 62580 atggaacgtt cttccatttg tttgtatcct cttttatttc attgagcagt ggtttgtagt 62640 tctccttgaa gaggtccttc acatctcttg taagttggat tcctaggtat tttattctct 62700 ttgaagcaat tgtgaatggg agttcactca tgatttggct ctctgtttgt ctgtcattgg 62760 tgtataagaa tgcttgtgat ttttgtacat tgattttgta tcctgagact tagctgaagt 62820 tgcttatcag cttgaggaga ttttgggctg agatgatggg gttttctaaa tatacaatca 62880 tgtcatctgc aaacagggac aatttgactt ctttttttcc taattgaatg ccctttattt 62940 ccttctcctg cctgattgcc ctggccagaa cttccaacac tatgttgaat aggagtggtg 63000 agagagggca tccctgtctt gtgccagttt tcaaagggaa tgcttccagt ttttgcccat 63060 tcagtatgat attggctgtg ggtttgtcat agatagctct tattattttg agatatgtcc 63120 catcaatacc taatttattg agagtgttta gcatgaaggg ttgttgaatt ttgtcaaagg 63180 ccttttctgc atctattgag ataatcatgt ggtttttgtc tttagttctg tttatacctt 63240 ggattacatt tattgatttg cgtatgttga actggctttg catcccaggg atgaagccca 63300 cttgatcatg gcggataagc tttttgatgt gctgctggat tcggtttgcc agtattttac 63360 tgaggatttt tgcatcgatg ttcatcaagg atattggcta aaattctctt tttttgttgt 63420 gtctctgcca ggctttggta tcaggatgat gctggcctca taaaatgagt tagggaggat 63480 tccctctttt tctgttgatt ggaatagttt cagaaggaat ggtaccattt cctccttgta 63540 cctctggtac aattcggctg tgaatccatc tggtcctgga ctctttttgg ttggtaagct 63600 attgattatt gcctcaattt cagatcctgt tattggtcta ttcagagatt cagcttcttc 63660 ctggtttagt cttgggagga tgtatgtgtc gaggaattta tccatttctt ctagattttc 63720 tagttgattt gcatagaggt gtttatagta ttctctgatg gtagtttgta tttctgtggg 63780 atcggtggtg atctcccctt tatcattttt gattgcgtct atttgattct tctctctttt 63840 cttctttatt agtcttgcta gcggtctatc aattttgttg atcctttcaa aaaaccagct 63900 cctggattca ttaatttttt gaagggtttt ttgtgtctct atttccttca gttctgctct 63960 gatcttagtt atttcttgcc ttctgctagc ttttgaatgt gtttgctctt gcttttctag 64020 ttcttttaat tgtgatgtaa gggtgtcaat tttagatctt tcctgctttc tcttgtgggc 64080 atttagtgct atacatttcc ctctacacac tgctttgaat gtgtcccaga gattctggta 64140 tgttgtcttt gttctcgttg gtttcaaaga acatctttat ttctgccttc atttcattat 64200 ttacccagta gtcattcggg agcaggttgt tcagtttcca tgtagttgag cagttttgag 64260 tgagtttctt aatcctgagt tctagtttga ttgcactgtg gtctgagaga cagtttgtta 64320 taatttctgt tcttttacat ttgctgagga gtgctttact tccaactatg tggtcaattt 64380 tggagtaggt gtggtgtggt gctgaaaaga atgtatattc tgttgatttg gggtggagag 64440 ttctgtagat gtctattagg tcctcttgtt gcagagctga gttcaattcc tgggtatcct 64500 tgttaacttt ctgtctcgtt gatctgtcta atgttgacag tggggtgtta aagtctccca 64560 ttattattgt gttggagtct aagtctcttt gtaggtcact aaggacttgc tttatgaatc 64620 tgggtgctcc tgtattgggt gcatatatat ttaggatagt tagctcttgt tgttgaattg 64680 atccctttac cattatgtaa tggccttctt tgtctctttt gatctttgtt ggtttaaagt 64740 ctgttttatc agagactagg attgcaatcc ctgccttctt ttggtttcca tttgcttggt 64800 agatcttcct ccatcccttt attttgagcc tatgtgtgtc tctgcacgtg agatgggttt 64860 cctgaataca gcacactgat gagtcctgac tctttatcca gtctgccagt ctgtgtcttt 64920 taattggagc atttagccca tttacgttta aagttaatat tgttatgtgt gaatttgatc 64980 ctgtcattat gatgttagct ggttattttg ctcgttagtt ggtgcagttt cttcctagcc 65040 ttgatggtct ttacaatttg gcatgttttt gcagtggctg gtactggttg ttcctttcca 65100 tgtttagtgc ttccttcagg agctcttgta aggcaggcct ggtggtgaca aaatctctca 65160 gcatttgctt gtctgtaaag gattttattt ctccttcact tatgaagctt agtttggctg 65220 gatatgaaat tctgggttga aaattctttt ctttaagaat gttgaatatt ggcccccact 65280 ctcttctggc ttgtagagtt tctgctgaga gatcagcttt tagtctgatg ggcttccctt 65340 tgtgggtaac ccgacctttc tctctggctg cccttaacat tttttccttc atttcaactt 65400 tggtgaatct gacaattatg tgtcttggag ttgctcttct cgaggagtat ctttgtggca 65460 ttctctgtat ttcctgaatt tgaatgttgg cctgccttgc tagattgggg aagttctcct 65520 ggataatata ctgcagagtg ttttccaact tggttccatt ctccccgtca ctttcaggta 65580 taccaatcag acgtagattt ggccttttca catagtccca tatttcttgg aggctttgtt 65640 cgtttctttt tattcttttt tctctaaact tctcttctcg cttcatttca ttcatttcgt 65700 cttccatcac tgataccctt tcttccagtt gatcgcatcg gctcctgagg cttctgcatt 65760 cgtcacgtag ctctcgtgcc ttggttttca gctccatcag gtcctttaag gacttgtctg 65820 cattgattat tcttgttatc cattcatctt attttttttc aaagctttta acttctttgc 65880 cattgattcg aatttcctcc tgtagctcag agtagtctga tcatctgagg ccttctttca 65940 actcgtcaga gtcattctcc atccagcttt gttccgttgc tggtgaggag ctgcgttcct 66000 ttggaggagg agaggcgctc tgatttttag agtttccagt ttttctgctc tgttttttcg 66060 agacaggatc tctctctgtt gcccaggctg gagtgcatgg cgcaatcatg gctcactgta 66120 gcctcaacct ccccaggttc tgctgatctt cccacttcag cctccagagt agctgggact 66180 ataggcgtgc accaccatac ctgcctaatt tttctatttt ttgtaaagac agggttttgc 66240 catgttgctc aggctgggct caaacgatct gcccaccaca gtctcccaaa gtgctggaat 66300 tacaggcatg gcatcagcca ccatgcccat ccatggaaca taagtttaga agataattag 66360 tgagtgtggc ttttggagtc tttacacttt attcattgga aatcctcaga taatggcaat 66420 gatactctac ttattgagag tgagattttg ccagtttgtg gggaaacccg gagtcagggg 66480 tttactaggc cttttatact cctgcctgcc taatcctgat aattttatat ggttgcaaaa 66540 attgccttta gcaggccgca tcatttgtat ttaacatata tgtttcaagt acaatttctt 66600 ataactagtg ttcttttatt gtattgccca ctagtgatct aatagtaagt atcacatgca 66660 actttatgaa ggcatgcact gtcatttgct ttcgtattat gaagcccaga agtgacagag 66720 cagggcaggg cagggcaggg catactgccc tgtgttaatt atgaatctct ttcctattgg 66780 attataacct ctattttgtt aatccctgag tctcatgttt tacctaggac ttgactcagg 66840 ataggtactc aaaatatatt tattgaattg aatgaaatta ctggaagaac ccacattgga 66900 atatcaccct ctggaacata cttagcatgt ctttgtaatt gaggttactg attatggcat 66960 actggcctat tagttatgag atctgggttc tgccaagtat aacccattag tctctagtat 67020 ctgttggaac taaaagtctc ttagtacaat gtaggacccc ttttgctgtt catgagagtt 67080 gtattctgga aactgtgggc actgggaaat gtgtaaatac tatatgctaa gcctgcgatt 67140 agtaaagata ccattcttaa ttaaatacca aattatagtt cttgaaaggt tagtgacctc 67200 taaagttttc ttcctgatat agtaatataa ctagtaataa atgggttata ttgaatcttt 67260 accatatttt aggcactaaa cactttatat atttaggcac taagcacttt atatattgtc 67320 ctttacaaca actctgtgag tcataggttc tattctcatt tttacaggtg agaaaactga 67380 ggtttcaagt gtttagtaac ttttccatga tagctggtaa gtagcagatc caggatttca 67440 gcctgtttgg ctctcaagtg tgtatgctct tggccactgc tcttaatagg ctcaaagagt 67500 atcacaactg gaagccctta gggctcatct agtctgtttt gtttattaga cagagaagga 67560 aactgaggcc tcccaattcg atgttggatc atgctgctct caacttaggg gaattgcaga 67620 aacatgtgat ctgagaaagc tggcttttta acttgttggt gtacattgta aatattgaaa 67680 accatgactt tgagaatcac aaatcataaa agcttcacat ctgtttctcc tgtgagaaca 67740 cacttataga cagaaataca tagaatgata tatattggaa ttcatatctt catgatttag 67800 ctaggatctt ttattccttt catagtaata gtacgttctt gttctcagta gctctttata 67860 ctatctctgt cccaacagaa gttgcagcaa ttctcagcaa agaatattag tatatatgtt 67920 tctctctctt tctctttctc tctctctctc tgtctctctc tctctctgtg tgtgtgtgtg 67980 tgtgtgtgtg tgtatacata tatacaatat gactaatcaa gggaattgta agtatggcag 68040 aagaatcaga tatgctcatg tgagttgagt agttatgttt agaggtcaaa tctggttctc 68100 aatttcctgg tagccagggc agaaaaggaa atgtgatagc tcctatattt tttatcaaat 68160 agaagaacat cctacagctt taagacacaa agctttttta ctactaaatg tttaaaaaga 68220 aaaaaaagca ccatttttaa gaggctctga gaattacata aggaattgtt agaccaggca 68280 cggtggctca tgcctgttat cccagcactt cgcgaggctg aggccagtgg atcagttgag 68340 gtcaggagtt tgagactagc ctggacagta tggcaaaacc ccgtctctac taaaaataca 68400 aaaattagcc aggtgtggtg gcgggcgcct gtagtcccag ctattcggga ggttgaggca 68460 ggagaattgc ttgaacccag gaggcggagg ttgcagtgag ccgagaccgc accactgcac 68520 tccagcctgg gcaacagagt gagactccgt ctcaaaaaaa aaaaaaaaaa aggaattgct 68580 ttctgtaaca gttttactga aaatttagat gcaattttca tataatattt tctcctaaat 68640 agtcattaaa tgttgaaggt atggcccaaa gtacagccca gtgaaagtga tttgggaaag 68700 gaccaagcta ctgaactttt tactgaatga ttagcattat tggatttgag gatagaacaa 68760 taaattccta gatcaatgct cctaaaatgc agtattgcga tatactactg ggtcatgatc 68820 tattaatacc ttccccagag gtaattatta ttctgattat caccatggtt aggcttacct 68880 cggaagaaat ggaataatac aatatgaact tgcttgtctg gcttctttcc cttaatgtgt 68940 ctgtgagatt caacccattt gtcagtgtac caattactta ttgtattgct gtgtaatttt 69000 ctgtatagta ttatatgaat ataacactat tcatttttcc attctcttgg acatttgggt 69060 tctttctggt tattggctat tataagcaaa tcttttatga acatttctgt gtatgtcttt 69120 tggtggctat acaaactaat ttctcttggg aatataccta ggattggaat tactagagta 69180 taggtagatg tatttatctg taagtagatg ctgccaaaca gttttccaaa gtggtctgtt 69240 ttaagatgtc cttcaatttg gggatgatca gaagctttcc acacagcacc ctttacaaac 69300 aaagatgcct ttatgtcttg taatggttac tggggaagaa tgttaaatag tcatgtgtct 69360 tttgctattt ttctcctttc tagcatcaat tcctgaagat tgccaagccc ctctccagcc 69420 tcactccact gattgctgca gctaaggagg caacaaagaa caatcactaa aaccacactc 69480 accccagcct cattgtgcca agccttctgt gagataaatg cacatttcag aaattccaac 69540 tcctgatgcc ctcttctcct tgccttgctt ctcccatttc ctgatctagc actcctcaag 69600 actttgatcc ttggaaaccg tgtgtccagc attgaagaga actgcaactg aatgactaat 69660 cagatgatgg ccatttctaa ataaggaatt tcctcccaat tcatggatat gagggtggtt 69720 tatgattaag ggtttatata aataaatgtt tctagtcttc cgtgtgtcaa aatcctcacc 69780 tccttcataa ccatctccca caattaattc ttgactatat aaatttatgg tttgataata 69840 ttatcaattt gtaatcaatt gagatttctt tagtgcttgc ttttctgtga ctcaactgcc 69900 cagacacctc attgtacttg aaaactggaa cagcttggga atgccatggg gtttgataat 69960 ctgccaggga catgaagagg ctcagcttcc tggaccatga ctttggctca gctgatcctg 70020 acatgggaga acaaccacat ttt 70043 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 tgtgattgaa ccacttcctg tca 23 <210> SEQ ID NO 6 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 ggagtggtgt tattttcagt aggtgaa 27 <210> SEQ ID NO 7 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 7 tccaactcgg gacgtggcta ca 22 <210> SEQ ID NO 8 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 9 gaagatggtg atgggatttc 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 10 caagcttccc gttctcagcc 20 <210> SEQ ID NO 11 <211> LENGTH: 547 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 165 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 386 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 427 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 458 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 473 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 476 <223> OTHER INFORMATION: unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 540 <223> OTHER INFORMATION: unknown <221> NAME/KEY: unsure <222> LOCATION: 543 <223> OTHER INFORMATION: unknown <223> OTHER INFORMATION: <400> SEQUENCE: 11 ggtttggcct cgctcccacg ggtcggtcag gccctccccg ccccctctca cctgcgggag 60 gcggggcggg cacggctcca ccggagccgc agccgccgcc gccgcgccgg ggagggggag 120 tgggggaggg ggagggggag aaggggaaag ggcagagggg aggangaggc gggaggagga 180 ggagtagggg agccagtgaa ctaggcggtc aggcctgccg ggcggcgtac aatagcgcgg 240 ctgtgggcgg gggaggctgc cctcccgcgg ctgcagccgg agccgaaggt ggtggctgca 300 cagtagacgc cccctcacgg cttcccccac acgctcccgc cccctcgctc gcccatcgcg 360 cttccctcac aggctctgca gtcctncccc acagacgcct tcccccttgg actctcattc 420 ccttttncac ggagccccgc gctttcgtga gccccctnga ggaacctggt ctncgnatcc 480 agttaccatc tcctgcctca taggccatct gagccctttg cacctcgccc ttaattcttn 540 aanaagc 547 <210> SEQ ID NO 12 <220> FEATURE: <400> SEQUENCE: 12 000 <210> SEQ ID NO 13 <211> LENGTH: 295 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 13 tcggagccca gcccagctga atcgagcgga ccggtggagc aggggccttg tgggtacccg 60 gttgggcagg gagaggtgcg gctctgcgac ggaaacaatc gccagagatg ccggggctag 120 ccttccccac cagtagctgc tgctggtggt gacaatgtca aataacggcc tagacattca 180 agacaaaccc ccagcccctc cgatgagaaa taccagcact atgattggag ccggcagcaa 240 agatgctgga accctaaacc atggttctaa acctctgcct ccaaacccag aggag 295 <210> SEQ ID NO 14 <211> LENGTH: 242 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 14 gccctcactc ccggctaggc ggcacccccc cgccgggagg aggaggagga gccgagagga 60 gctgagcgag cgcggaagta gctgctgctg gtggtgacaa tgtcaaatac cagcactatg 120 attggagccg gcagcaaaga tgctggaacc ctaaaccatg gttctaaacc tctgcctcca 180 aacccagagg agaagaaaaa gaaggaccga ttttaccgat ccattttacc tggagataaa 240 ac 242 <210> SEQ ID NO 15 <211> LENGTH: 2318 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (394)...(2031) <400> SEQUENCE: 15 gccacgaagg ccacagacgc cttccccctt ggactctcat tcccttttcc acggagcccc 60 gcgctttcgt gagccccctc gaggaacctg gtctccgcat ccagttacca cctcctgcct 120 cagaggccat ctgagccctt cgcacctcgc ccctcagtcc ccccttgccc ccccgcggag 180 atcgcctcgc tccctcccgc ccccccatca tcccttccct cgcagttccc ctgtcctgag 240 gggagccccg ccacggcagc gacagcgggc aggagggaga aagtgaaggt tgggcgacac 300 ttggcctcac tcccggctag gcgcacccac ggggaggaga ggaggagccg agagagctga 360 gcagcgcgga agtagctgct gctggtggtg aca atg tca aat aac ggc cta gac 414 Met Ser Asn Asn Gly Leu Asp 1 5 att caa gac aaa ccc cca gcc cct ccg atg aga aat acc agc act atg 462 Ile Gln Asp Lys Pro Pro Ala Pro Pro Met Arg Asn Thr Ser Thr Met 10 15 20 att gga gtc ggc agc aaa gat gct gga acc cta aac cat ggt tct aaa 510 Ile Gly Val Gly Ser Lys Asp Ala Gly Thr Leu Asn His Gly Ser Lys 25 30 35 cct ctg cct cca aac cca gag gag aag aaa aag aag gac cga ttt tac 558 Pro Leu Pro Pro Asn Pro Glu Glu Lys Lys Lys Lys Asp Arg Phe Tyr 40 45 50 55 cga tcc att tta cct gga gat aaa aca aat aaa aag aaa gag aaa gag 606 Arg Ser Ile Leu Pro Gly Asp Lys Thr Asn Lys Lys Lys Glu Lys Glu 60 65 70 cgg cca gag att tct ctc cct tca gat ttt gaa cac aca att cat gtc 654 Arg Pro Glu Ile Ser Leu Pro Ser Asp Phe Glu His Thr Ile His Val 75 80 85 ggt ttt gat gct gtc aca ggg gag ttt acg gga atg cca gag cag tgg 702 Gly Phe Asp Ala Val Thr Gly Glu Phe Thr Gly Met Pro Glu Gln Trp 90 95 100 gcc cgc ttg ctt cag aca tca aat atc act aag tcg gag cag aag aaa 750 Ala Arg Leu Leu Gln Thr Ser Asn Ile Thr Lys Ser Glu Gln Lys Lys 105 110 115 aac ccg cag gct gtt ctg gat gtg ttg gag ttt tac aac tcg aag aag 798 Asn Pro Gln Ala Val Leu Asp Val Leu Glu Phe Tyr Asn Ser Lys Lys 120 125 130 135 aca tcc aac agc cag aaa tac atg agc ttt aca gat aag tca gct gag 846 Thr Ser Asn Ser Gln Lys Tyr Met Ser Phe Thr Asp Lys Ser Ala Glu 140 145 150 gat tac aat tct tct aat gcc ttg aat gtg aag gct gtg tct gag act 894 Asp Tyr Asn Ser Ser Asn Ala Leu Asn Val Lys Ala Val Ser Glu Thr 155 160 165 cct gca gtg cca cca gtt tca gaa gat gag gat gat gat gat gat gat 942 Pro Ala Val Pro Pro Val Ser Glu Asp Glu Asp Asp Asp Asp Asp Asp 170 175 180 gct acc cca cca cca gtg att gct cca cgc cca gag cac aca aaa tct 990 Ala Thr Pro Pro Pro Val Ile Ala Pro Arg Pro Glu His Thr Lys Ser 185 190 195 gta tac aca cgg tct gtg att gaa cca ctt cct gtc act cca act cgg 1038 Val Tyr Thr Arg Ser Val Ile Glu Pro Leu Pro Val Thr Pro Thr Arg 200 205 210 215 gac gtg gct aca tct ccc att tca cct act gaa aat aac acc act cca 1086 Asp Val Ala Thr Ser Pro Ile Ser Pro Thr Glu Asn Asn Thr Thr Pro 220 225 230 cca gat gct ttg acc cgg aat act gag aag cag aag aag aag cct aaa 1134 Pro Asp Ala Leu Thr Arg Asn Thr Glu Lys Gln Lys Lys Lys Pro Lys 235 240 245 atg tct gat gag gag atc ttg gag aaa tta cga agc ata gtg agt gtg 1182 Met Ser Asp Glu Glu Ile Leu Glu Lys Leu Arg Ser Ile Val Ser Val 250 255 260 ggc gat cct aag aag aaa tat aca cgg ttt gag aag att gga caa ggt 1230 Gly Asp Pro Lys Lys Lys Tyr Thr Arg Phe Glu Lys Ile Gly Gln Gly 265 270 275 gct tca ggc acc gtg tac aca gca atg gat gtg gcc aca gga cag gag 1278 Ala Ser Gly Thr Val Tyr Thr Ala Met Asp Val Ala Thr Gly Gln Glu 280 285 290 295 gtg gcc att aag cag atg aat ctt cag cag cag ccc aag aaa gag ctg 1326 Val Ala Ile Lys Gln Met Asn Leu Gln Gln Gln Pro Lys Lys Glu Leu 300 305 310 att att aat gag atc ctg gtc atg agg gaa aac aag aac cca aac att 1374 Ile Ile Asn Glu Ile Leu Val Met Arg Glu Asn Lys Asn Pro Asn Ile 315 320 325 gtg aat tac ttg gac agt tac ctc gtg gga gat gag ctg tgg gtt gtt 1422 Val Asn Tyr Leu Asp Ser Tyr Leu Val Gly Asp Glu Leu Trp Val Val 330 335 340 atg gaa tac ttg gct gga ggc tcc ttg aca gat gtg gtg aca gaa act 1470 Met Glu Tyr Leu Ala Gly Gly Ser Leu Thr Asp Val Val Thr Glu Thr 345 350 355 tgc atg gat gaa ggc caa att gca gct gtg tgc cgt gag tgt ctg cag 1518 Cys Met Asp Glu Gly Gln Ile Ala Ala Val Cys Arg Glu Cys Leu Gln 360 365 370 375 gct ctg gag ttc ttg cat tcg aac cag gtc att cac aga gac atc aag 1566 Ala Leu Glu Phe Leu His Ser Asn Gln Val Ile His Arg Asp Ile Lys 380 385 390 agt gac aat att ctg ttg gga atg gat ggc tct gtc aag cta act gac 1614 Ser Asp Asn Ile Leu Leu Gly Met Asp Gly Ser Val Lys Leu Thr Asp 395 400 405 ttt gga ttc tgt gca cag ata acc cca gag cag agc aaa cgg agc acc 1662 Phe Gly Phe Cys Ala Gln Ile Thr Pro Glu Gln Ser Lys Arg Ser Thr 410 415 420 atg gta gga acc cca tac tgg atg gca cca gag gtt gtg aca cga aag 1710 Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Thr Arg Lys 425 430 435 gcc tat ggg ccc aag gtt gac atc tgg tcc ctg ggc atc atg gcc atc 1758 Ala Tyr Gly Pro Lys Val Asp Ile Trp Ser Leu Gly Ile Met Ala Ile 440 445 450 455 gaa atg att gaa ggg gag cct cca tac ctc aat gaa aac cct ctg aga 1806 Glu Met Ile Glu Gly Glu Pro Pro Tyr Leu Asn Glu Asn Pro Leu Arg 460 465 470 gcc ttg tac ctc att gcc acc aat ggg acc cca gaa ctt cag aac cca 1854 Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Glu Leu Gln Asn Pro 475 480 485 gag aag ctg tca gct atc ttc cgg gac ttt ctg aac cgc tgt ctc gat 1902 Glu Lys Leu Ser Ala Ile Phe Arg Asp Phe Leu Asn Arg Cys Leu Asp 490 495 500 atg gat gtg gag aag aga ggt tca gct aaa gag ctg cta cag cat caa 1950 Met Asp Val Glu Lys Arg Gly Ser Ala Lys Glu Leu Leu Gln His Gln 505 510 515 ttc ctg aag att gcc aag ccc ctc tcc agc ctc act cca ctg att gct 1998 Phe Leu Lys Ile Ala Lys Pro Leu Ser Ser Leu Thr Pro Leu Ile Ala 520 525 530 535 gca gct aag gag gca aca aag aac aat cac taa aaccacactc accccagcct 2051 Ala Ala Lys Glu Ala Thr Lys Asn Asn His 540 545 cattgtgcca agctctgtga gataaatgca catttcagaa attccaactc ctgatgccct 2111 cttctccttg ccttgcttct cccatttcct gatctagcac tcctcaagac tttgatcctt 2171 ggaaaccgtg tgtccagcat tgaagagaac tgcaactgaa tgactaatca gatgatggcc 2231 atttctaaat aaggaatttc ctcccaattc atggatatga gggtggttta tgattaaggg 2291 tttatataaa taaatgtttc tagtctt 2318 <210> SEQ ID NO 16 <220> FEATURE: <400> SEQUENCE: 16 000 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 17 atggcctctg aggcaggagg 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 18 ttgtcaccac cagcagcagc 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 19 tgacattgtc accaccagca 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 20 ttatttgaca ttgtcaccac 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 21 ctctgggttt ggaggcagag 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 22 ttctcctctg ggtttggagg 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 23 aaactcccct gtgacagcat 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 cccgtaaact cccctgtgac 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 gcattcccgt aaactcccct 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 tgaagcaagc gggcccactg 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 caacacatcc agaacagcct 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 tcagctgact tatctgtaaa 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 ccttcacatt caaggcatta 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 gggcgtggag caatcactgg 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 ggaagtggtt caatcacaga 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 ttccgggtca aagcatctgg 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 cagtattccg ggtcaaagca 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 agacatttta ggcttcttct 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 ctcatcagac attttaggct 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 36 atcgcccaca ctcactatgc 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 37 ttaggatcgc ccacactcac 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 38 tcttcttagg atcgcccaca 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 39 attaataatc agctctttct 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 40 gatctcatta ataatcagct 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 41 cacgaggtaa ctgtccaagt 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 42 aacaacccac agctcatctc 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 43 ttccataaca acccacagct 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 tgtcaaggag cctccagcca 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 acatctgtca aggagcctcc 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 tcaccacatc tgtcaaggag 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 gaaagtcccg gaagatagct 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 agctgaacct ctcttctcca 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 ctctttagct gaacctctct 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 atcttcagga attgatgctg 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 ggcttggcaa tcttcaggaa 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 gctgcagcaa tcagtggagt 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 agagcttggc acaatgaggc 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 aggagttgga atttctgaaa 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 aggaaatggg agaagcaagg 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 agatcaggaa atgggagaag 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 gagtgctaga tcaggaaatg 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 agtcttgagg agtgctagat 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 ttccaaggat caaagtcttg 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 tgctggacac acggtttcca 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 aaatggccat catctgatta 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 cttatttaga aatggccatc 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 attgggagga aattccttat 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 ccctcatatc catgaattgg 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 ctagaaacat ttatttatat 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 accgcctagt tcactggctc 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 aggcctgacc gcctagttca 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 agagcctgtg agggaagcgc 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 tatagtcaag aattaattgt 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 ccagcagcag ctactggtgg 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 tagtgctggt atttgacatt 20 <210> SEQ ID NO 72 <220> FEATURE: <400> SEQUENCE: 72 000 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 gaaatctcaa ttgattacaa 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 aaccccatgg cattcccaag 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 ggaagctgag cctcttcatg 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 ctgagccaaa gtcatggtcc 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 ttctcccatg tcaggatcag 20 <210> SEQ ID NO 78 <220> FEATURE: <400> SEQUENCE: 78 000 <210> SEQ ID NO 79 <220> FEATURE: <400> SEQUENCE: 79 000 <210> SEQ ID NO 80 <220> FEATURE: <400> SEQUENCE: 80 000 <210> SEQ ID NO 81 <220> FEATURE: <400> SEQUENCE: 81 000 <210> SEQ ID NO 82 <220> FEATURE: <400> SEQUENCE: 82 000 <210> SEQ ID NO 83 <220> FEATURE: <400> SEQUENCE: 83 000 <210> SEQ ID NO 84 <220> FEATURE: <400> SEQUENCE: 84 000 <210> SEQ ID NO 85 <220> FEATURE: <400> SEQUENCE: 85 000 <210> SEQ ID NO 86 <220> FEATURE: <400> SEQUENCE: 86 000 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 87 ttctgacttc aggtgatcca 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 88 cacaattatc ttttgcatag 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 89 actatgcttc ctttgaaaga 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 90 catatagcct aggtgtgtag 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 91 gcctgaagca ctgaacagta 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 92 taatggccac ctgaaatcaa 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 93 ctcaaatgaa acatctagtt 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 94 tcctacttac ttagcttgac 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 95 cctcctgcct cagaggccat 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 96 gctgctgctg gtggtgacaa 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 97 tgctggtggt gacaatgtca 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 98 gtggtgacaa tgtcaaataa 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 99 ctctgcctcc aaacccagag 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 100 cctccaaacc cagaggagaa 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 101 atgctgtcac aggggagttt 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 102 gtcacagggg agtttacggg 20 <210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 103 aggggagttt acgggaatgc 20 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 104 cagtgggccc gcttgcttca 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 105 aggctgttct ggatgtgttg 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 106 tttacagata agtcagctga 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 107 taatgccttg aatgtgaagg 20 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 108 ccagtgattg ctccacgccc 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 109 tctgtgattg aaccacttcc 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 110 ccagatgctt tgacccggaa 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 111 tgctttgacc cggaatactg 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 112 agaagaagcc taaaatgtct 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 113 agcctaaaat gtctgatgag 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 114 gcatagtgag tgtgggcgat 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 115 gtgagtgtgg gcgatcctaa 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 116 tgtgggcgat cctaagaaga 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 117 agaaagagct gattattaat 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 118 agctgattat taatgagatc 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 119 acttggacag ttacctcgtg 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 120 gagatgagct gtgggttgtt 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 121 agctgtgggt tgttatggaa 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 122 tggctggagg ctccttgaca 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 123 ggaggctcct tgacagatgt 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 124 ctccttgaca gatgtggtga 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 125 agctatcttc cgggactttc 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 126 tggagaagag aggttcagct 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 127 agagaggttc agctaaagag 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 128 cagcatcaat tcctgaagat 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 129 ttcctgaaga ttgccaagcc 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 130 actccactga ttgctgcagc 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 131 gcctcattgt gccaagctct 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 132 tttcagaaat tccaactcct 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 133 ccttgcttct cccatttcct 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 134 cttctcccat ttcctgatct 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 135 catttcctga tctagcactc 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 136 atctagcact cctcaagact 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 137 caagactttg atccttggaa 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 138 tggaaaccgt gtgtccagca 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 139 taatcagatg atggccattt 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 140 gatggccatt tctaaataag 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 141 ataaggaatt tcctcccaat 20 <210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 142 ccaattcatg gatatgaggg 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 143 atataaataa atgtttctag 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 144 ccaccagtag ctgctgctgg 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 145 aatgtcaaat accagcacta 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 146 ccagagatgc cggggctagc 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 147 ttgtaatcaa ttgagatttc 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 148 cttgggaatg ccatggggtt 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 149 catgaagagg ctcagcttcc 20 <210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 150 ggaccatgac tttggctcag 20 <210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 151 ctgatcctga catgggagaa 20 <210> SEQ ID NO 152 <220> FEATURE: <400> SEQUENCE: 152 000 <210> SEQ ID NO 153 <220> FEATURE: <400> SEQUENCE: 153 000 <210> SEQ ID NO 154 <220> FEATURE: <400> SEQUENCE: 154 000 <210> SEQ ID NO 155 <220> FEATURE: <400> SEQUENCE: 155 000 <210> SEQ ID NO 156 <220> FEATURE: <400> SEQUENCE: 156 000 <210> SEQ ID NO 157 <220> FEATURE: <400> SEQUENCE: 157 000 <210> SEQ ID NO 158 <220> FEATURE: <400> SEQUENCE: 158 000 <210> SEQ ID NO 159 <220> FEATURE: <400> SEQUENCE: 159 000 <210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 160 tggatcacct gaagtcagaa 20 <210> SEQ ID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 161 ctatgcaaaa gataattgtg 20 <210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 162 tctttcaaag gaagcatagt 20 <210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 163 ctacacacct aggctatatg 20 <210> SEQ ID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 164 tactgttcag tgcttcaggc 20 <210> SEQ ID NO 165 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 165 ttgatttcag gtggccatta 20 <210> SEQ ID NO 166 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 166 aactagatgt ttcatttgag 20 <210> SEQ ID NO 167 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 167 gtcaagctaa gtaagtagga 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding PAK1, wherein said compound specifically hybridizes with said nucleic acid molecule encoding PAK1 (SEQ ID NO: 4) and inhibits the expression of PAK1.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding PAK1 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PAK1.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding PAK1 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PAK1.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding PAK1 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PAK1.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding PAK1 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PAK1.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of PAK1 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of PAK1 is inhibited.
 19. A method of screening for a modulator of PAK1, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding PAK1 with one or more candidate modulators of PAK1, and b. identifying one or more modulators of PAK1 expression which modulate the expression of PAK1.
 20. The method of claim 19 wherein the modulator of PAK1 expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of PAK1 in a sample using at least one of the primers comprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with PAK1 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of PAK1 is inhibited.
 24. The method of claim 23 wherein the disease or condition is a hyperproliferative disorder. 